Polymerization of isoprene from renewable resources

ABSTRACT

This invention relates to compositions and methods for producing polymers of isoprene derived from renewable resources, such as isoprene produced from cultured cells that use renewable carbon sources. A starting isoprene composition, such as a bioisoprene composition, is distinguished from petroleum based isoprene by its purity profile (such as lower levels of certain C 5  hydrocarbons other than isoprene, presence of certain compounds associated with the biological process for production) and the relative content of the carbon isotopes. Polymers obtained by polymerization of such starting isoprene composition according to this invention, such as a polyisoprene homopolymer or a copolymer having repeat units that are derived from isoprene, are distinguishable from isoprene containing polymers from petrochemical resources.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.12/817,093, filed on Jun. 16, 2010, (now granted as U.S. Pat. No.8,546,506 on Oct. 1, 2013) which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 61/187,944, filed Jun. 17, 2009. Theteachings of U.S. patent application Ser. No. 12/817,093 (U.S. Pat. No.8,546,506) and U.S. Provisional Patent Application Ser. No. 61/187,944are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Isoprene (2-methyl-buta-1,3-diene) is an important organic compound thatis used in a wide array of applications. For instance, isoprene isemployed as an intermediate or a starting material in the synthesis ofnumerous chemical compositions and polymers. Isoprene is also animportant biological material that is synthesized naturally by manyplants and animals, including humans. Isoprene is a colorless liquid atroom temperature and is highly flammable.

Isoprene became an important monomer for utilization in the synthesis ofcis-1,4-polybutadiene when its stereo-regulated polymerization becamecommercially possible in the early 1960s. cis-1,4-Polyisoprene made bysuch stereo-regulated polymerizations is similar in structure andproperties to natural rubber. Even though it is not identical to naturalrubber it can be used as a substitute for natural rubber in manyapplications. For instance, synthetic cis-1,4-polyisoprene rubber iswidely used in manufacturing tires and other rubber products. Thisdemand for synthetic cis-1,4-polyisoprene rubber consumes a majority ofthe isoprene available in the worldwide market. The remaining isopreneis used in making other synthetic rubbers, block copolymers, and otherchemical products. For instance, isoprene is used in makingbutadiene-isoprene rubbers, styrene-isoprene copolymer rubbers,styrene-isoprene-butadiene rubbers, styrene-isoprene-styrene blockcopolymers, and styrene-isoprene block copolymers.

Over the years many synthesis routes for producing isoprene have beeninvestigated. For instance, the synthesis of isoprene by reactingisobutylene with formaldehyde in the presence of a catalyst is describedin U.S. Pat. No. 3,146,278, U.S. Pat. No. 3,437,711, U.S. Pat. No.3,621,072, U.S. Pat. No. 3,662,016, U.S. Pat. No. 3,972,955, U.S. Pat.No. 4,000,209, U.S. Pat. No. 4,014,952, U.S. Pat. No. 4,067,923, andU.S. Pat. No. 4,511,751. U.S. Pat. No. 3,574,780 discloses anotherprocess for the manufacture of isoprene by passing a mixture ofmethyl-tert-butyl ether and air over mixed oxide catalysts. Themethyl-tert-butyl ether is then cracked into isobutylene and methanolover the catalyst. The methanol produced is oxidized into formaldehydewhich then reacts with the isobutylene over the same catalyst to producethe isoprene. U.S. Pat. No. 5,177,290 discloses a process for producingdienes, including isoprene, which involves reacting a reaction mixtureof a tertiary alkyl ether and a source of oxygen over two functionallydistinct catalysts under reaction conditions sufficient to produce highyields of the dienes with minimal recycle of the ether.

The isoprene used in industrial applications is typically produced as aby-product of the thermal cracking of petroleum or naphtha or isotherwise extracted from petrochemical streams. This is a relativelyexpensive energy-intensive process. With the worldwide demand forpetrochemical based products constantly increasing, the cost of isopreneis expected to rise to much higher levels in the long-term and itsavailability is limited in any case. In other words, there is a concernthat future supplies of isoprene from petrochemical based sources willbe inadequate to meet projected needs and that prices will rise tounprecedented levels. Accordingly, there is a current need to procure asource of isoprene from a low cost, renewable source which isenvironmentally friendly.

In addition, isoprene produced from petrochemical feedstocks requiresextensive purification before it can be converted to polymers. Costeffective methods are desirable for producing highly pure isoprene fromrenewable resources and converting it to polyisoprene products takingadvantage of the high purity and/or the unique impurity profiles ofbioisoprene compositions.

The invention described herein fulfills these needs and providesadditional benefits as well.

BRIEF SUMMARY OF THE INVENTION

The invention provides, inter alia, compositions and methods forproducing polymers of isoprene from renewable resources.

Accordingly, in one aspect, the invention provides systems for producinga copolymer of isoprene comprising: (a) an isoprene starting compositionderived from renewable resources; and (b) a polymer produced from atleast a portion of the isoprene starting material; wherein at least aportion of the isoprene starting composition undergoes polymerizationwith another non-isoprene molecule to produce a copolymer. In oneembodiment, the isoprene starting composition derived from renewableresources comprises greater than about 2 mg of isoprene and comprisesgreater than or about 99.94% isoprene by weight compared to the totalweight of all C5 hydrocarbons in the composition. In another embodiment,the isoprene starting composition derived from renewable resourcescomprises greater than about 2 mg of isoprene and comprises one or morecompounds selected from the group consisting of ethanol, acetone, C5prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.In another embodiment, the isoprene starting composition derived fromrenewable resources comprises greater than about 2 mg of isoprene andcomprises one or more second compounds selected from the groupconsisting of ethanol, acetone, methanol, acetaldehyde, methacrolein,methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol, 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,and 2,3-cycloheptenolpyridine; wherein the amount of the second compoundrelative to the amount of the isoprene is greater than or about 0.01%(w/w). In another embodiment, the isoprene starting composition derivedfrom renewable resources comprises greater than about 2 mg of isopreneand comprises less than or about 0.5 μg/L per compound for any compoundin the composition that inhibits the polymerization of isoprene. Inanother embodiment, the polymer produced from the isoprene startingmaterial is a polyisoprene polymer which is comprised of repeat unitsthat are derived from isoprene monomer, wherein the polyisoprene polymerhas δ¹³C value of greater than −22‰ or which is within the range of −30‰to −28.5‰. In another embodiment, the polymer produced from the isoprenestarting material is a polymer which is comprised of repeat units thatare derived from isoprene monomer and at least one additional monomer,wherein the polymer includes blocks of repeat units that are derivedfrom isoprene, and wherein the blocks of repeat units that are derivedfrom isoprene have a δ¹³C value of greater than −22‰ or which is withinthe range of −32‰ to −24‰. In another embodiment, the polymer producedfrom the isoprene starting material is a polymer which is comprised ofrepeat units that are derived from isoprene monomer and at least oneadditional monomer, wherein the polymer includes blocks of repeat unitsthat are derived from isoprene, and wherein the blocks of repeat unitsthat are derived from isoprene have a δ¹³C value of greater than −22‰ orwhich is within the range of −34‰ to −24‰. In another embodiment, thepolymer is a copolymer selected from the group consisted of (i)copolymers of isoprene and 1,3-butadiene, (ii) copolymers of isopreneand styrene, (iii) copolymers of isoprene, 1,3-butadiene, and styrene,and (iv) copolymers of isoprene and α-methyl styrene. In anotherembodiment, the polymer produced from the isoprene starting material isa polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer hasf_(M) value which is greater than 0.9. In another embodiment, the systemfurther comprises one or more of: (i) a catalyst for polymerizingisoprene, (ii) a polymerization initiator, (iii) an ionic surfactant,(iv) a suitable organic solvent, and (v) a polymerization chainterminator.

In another aspect, the invention provides for systems for producing apolymer of isoprene comprising: (a) an isoprene starting compositionderived from renewable resources; and (b) a polymer produced from atleast a portion of the isoprene starting material; wherein at least aportion of the isoprene starting composition undergoes polymerizationwith other isoprene molecules to produce a polymer of isoprene with amolecular weight of about 5,000 to about 100,000.

In another aspect, the invention provides for methods for producing acopolymer of isoprene derived from renewable resources comprising: (a)culturing cells comprising a heterologous nucleic acid encoding anisoprene synthase polypeptide under suitable culture conditions for theproduction of the isoprene; (b) producing the isoprene; and (c)polymerizing the isoprene derived from renewable resources with anothernon-isoprene molecules to produce a copolymer. In one embodiment, themethod further comprises recovering the isoprene from theisoprene-producing cell culture prior to polymerization. In anotherembodiment, the method further comprises step (d) recovering the polymerproduced.

In another aspect, the invention provides for methods for producing apolymer of isoprene derived from renewable resources comprising: (a)culturing cells comprising a heterologous nucleic acid encoding anisoprene synthase polypeptide under suitable culture conditions for theproduction of the isoprene; (b) producing the isoprene; and (c)polymerizing the isoprene derived from renewable resources with otherisoprene molecules to produce a polymer of isoprene with a molecularweight of about 5,000 to about 100,000.

In another aspect, the invention provides for polymers of isoprenederived from renewable resources produced by any of the methodsdescribed herein.

In one aspect, the invention provides for a system for producing apolymer of isoprene comprising: (a) an isoprene starting compositionderived from renewable resources; and (b) a polymer produced from atleast a portion of the isoprene starting material; where at least aportion of the isoprene starting composition undergoes polymerization.In some embodiments, the isoprene starting composition derived fromrenewable resources comprises greater than about 2 mg of isoprene andcomprises greater than or about 99.94% isoprene by weight compared tothe total weight of all C5 hydrocarbons in the composition. In someembodiments, the isoprene starting composition derived from renewableresources comprises greater than about 2 mg of isoprene and comprisesone or more compounds selected from the group consisting of ethanol,acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or morecarbon atoms. In some embodiments, the isoprene starting compositionderived from renewable resources comprises greater than about 2 mg ofisoprene and comprises one or more second compounds selected from thegroup consisting of ethanol, acetone, methanol, acetaldehyde,methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol, 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,citronellol and geraniol; wherein the amount of the second compoundrelative to the amount of the isoprene is greater than or about 0.01%(w/w). In some embodiments, the isoprene starting composition derivedfrom renewable resources comprises greater than about 2 mg of isopreneand comprises less than or about 0.5 μg/L per compound for any compoundin the composition that inhibits the polymerization of isoprene.

In some embodiments, the polymer produced from the isoprene startingmaterial is a polyisoprene polymer which is comprised of repeat unitsthat are derived from isoprene monomer, wherein the polyisoprene polymerhas δ13C value of greater than −22‰ or which is within the range of −30‰to −28.5‰. In some embodiments, the polymer produced from the isoprenestarting material is a polymer which is comprised of repeat units thatare derived from isoprene monomer and at least one additional monomer,wherein the polymer includes blocks of repeat units that are derivedfrom isoprene, and wherein the blocks of repeat units that are derivedfrom isoprene have a δ¹³C value of greater than −22‰ or which is withinthe range of −32‰ to −24‰. In some embodiments, the polymer producedfrom the isoprene starting material is a polymer which is comprised ofrepeat units that are derived from isoprene monomer and at least oneadditional monomer, wherein the polymer includes blocks of repeat unitsthat are derived from isoprene, and wherein the blocks of repeat unitsthat are derived from isoprene have a δ13C value of greater than −22‰ orwhich is within the range of −31‰ to −24‰. In some embodiments, thepolymer produced from the isoprene starting material is a polymer whichis comprised of repeat units that are derived from isoprene monomer andat least one additional monomer, wherein the polymer includes blocks ofrepeat units that are derived from isoprene, and wherein the blocks ofrepeat units that are derived from isoprene have a δ¹³C value of greaterthan −22‰ or which is within the range of −34‰ to −24‰. In someembodiments, the polymer is a copolymer selected from the groupconsisting of (i) copolymers of isoprene and 1,3-butadiene, (ii)copolymers of isoprene and styrene, (iii) copolymers of isoprene,1,3-butadiene, and styrene, and (iv) copolymers of isoprene and α-methylstyrene. In some embodiments, the polymer produced from the isoprenestarting material is a polyisoprene polymer which is comprised of repeatunits that are derived from isoprene monomer, wherein the polyisoprenepolymer has f_(M) value which is greater than 0.9.

In some embodiments, the system further comprises a catalyst forpolymerizing isoprene. In some embodiments, the system further comprisesa polymerization initiator. In some embodiments, the system furthercomprises an ionic surfactant. In some embodiments, the system furthercomprises a suitable organic solvent. In some embodiments, the systemfurther comprises a polymerization chain terminator. In someembodiments, the system further comprises one additional monomerselected from the group consisting of 1,3-butadiene and styrene. In someembodiments, the system further comprises additional monomers includingboth 1,3-butadiene and styrene.

In one aspect, provided is a method for producing a polymer of isoprenederived from renewable resources comprising: (a) obtaining isoprene fromrenewable resources; (b) polymerizing isoprene derived from renewableresources; and (c) recovering the polymer produced. In some embodiments,the isoprene from renewable resources is obtained by a method whichcomprises the steps of (i) culturing cells comprising a heterologousnucleic acid encoding an isoprene synthase polypeptide under suitableculture conditions for the production of the isoprene, (ii) producingthe isoprene, and (iii) recovering the isoprene from the culture. Apolymer of isoprene derived from renewable resources produced by any ofthe methods described herein is also provided.

In one aspect, provided is a polyisoprene polymer which is comprised ofrepeat units that are derived from isoprene monomer, wherein thepolyisoprene polymer has δ¹³C value of greater than −22‰. In someembodiments, the polyisoprene polymer has δ¹³C value which is within therange of −30‰ to −28.5‰. In some embodiments, the polyisoprene polymerhas δ¹³C value which is within the range of −32‰ to −24‰. In someembodiments, the polyisoprene polymer has δ¹³C value which is within therange of −34‰ to −24‰. In various embodiments, the polyisoprene is freeof protein. In some embodiments, the polyisoprene is a polyisoprenehomopolymer.

In one aspect, provided is a polyisoprene polymer which is comprised ofrepeat units that are derived from isoprene monomer, wherein thepolyisoprene polymer has a cis-1,4-microstructure content of less than99.9%, wherein the polyisoprene polymer has a trans-1,4-microstructurecontent of less than 99.9%, and wherein the polyisoprene polymer hasδ¹³C value of greater than −22‰. In some embodiments, the polyisoprenepolymer has δ¹³C value which is within the range of −30‰ to −28.5‰. Insome embodiments, the polyisoprene polymer has δ¹³C value which iswithin the range of −32‰ to −24‰. In some embodiments, the polyisoprenepolymer has δ¹³C value which is within the range of −34‰ to −24‰.

In one aspect, provided is a polyisoprene polymer which is comprised ofrepeat units that are derived from isoprene monomer, wherein thepolyisoprene polymer has a 3,4-microstructure content of greater than2%, and wherein the polyisoprene polymer has δ¹³C value of greater than−22‰. In some embodiments, the polyisoprene polymer has δ¹³C value whichis within the range of −30‰ to −28.5‰. In some embodiments, thepolyisoprene polymer has δ¹³C value which is within the range of −32‰ to−24‰. In some embodiments, the polyisoprene polymer has δ¹³C value whichis within the range of −34‰ to −24‰.

In one aspect, provided is a polyisoprene polymer which is comprised ofrepeat units that are derived from isoprene monomer, wherein thepolyisoprene polymer has a 1,2-microstructure content of greater than2%, and wherein the polyisoprene polymer has δ¹³C value of greater than−22‰. In some embodiments, the polyisoprene polymer has δ¹³C value whichis within the range of −30‰ to −28.5‰. In some embodiments, thepolyisoprene polymer has δ¹³C value which is within the range of −32‰ to−24‰. In some embodiments, the polyisoprene polymer has δ¹³C value whichis within the range of −34‰ to −24‰.

In one aspect, provided is a polymer which is comprised of repeat unitsthat are derived from isoprene monomer and at least one additionalmonomer, wherein the polymer includes blocks of repeat units that arederived from isoprene, and wherein the blocks of repeat units that arederived from isoprene have a δ¹³C value of greater than −22‰. In someembodiments, the polyisoprene polymer has δ¹³C value which is within therange of −30‰ to −28.5‰. In some embodiments, the polyisoprene polymerhas δ¹³C value which is within the range of −32‰ to −24‰. In someembodiments, the polyisoprene polymer has δ¹³C value which is within therange of −34‰ to −24‰.

In one aspect, provided is a liquid polyisoprene polymer which iscomprised of repeat units that are derived from isoprene monomer,wherein the liquid polyisoprene polymer has a weight average molecularweight which is within the range of 5,000 to 100,000, and wherein theliquid polyisoprene polymer has δ¹³C value of greater than −22‰. In someembodiments, the polyisoprene polymer has δ¹³C value which is within therange of −30‰ to −28.5‰. In some embodiments, the polyisoprene polymerhas δ¹³C value which is within the range of −32‰ to −24‰. In someembodiments, the polyisoprene polymer has δ¹³C value which is within therange of −34‰ to −24‰.

In one aspect, provided is a method for verifying that a polyisoprenehomopolymer is from a sustainable renewable non-petroleum derived sourcewhich comprises: (I) determining the δ¹³C value of the polyisoprenehomopolymer; (II) if the polyisoprene homopolymer has a δ¹³C valuewithin the range of −34‰ to −30‰ or within the range of −28.5‰ to −24‰additionally analyzing the polyisoprene homopolymer to determine (1) itscis-microstructure content, (2) its 3,4-microstructure content, (3) its1,2-microstructure content, (4) its a weight average molecular weight,or (5) the presence or absence of residual proteins, soaps, lipids,resins, or sugars indicative of natural rubber; and (III) verifying thatthe polyisoprene homopolymer is from a sustainable renewablenon-petroleum derived source if it has (i) a δ¹³C value of greater than−22‰, (ii) a δ¹³C value which is within the range of −30‰ to −28.5‰, or(iii) a δ¹³C value within the range of −34‰ to −30‰ or within the rangeof −28.5‰ to −24‰ and if it (a) has a cis-microstructure content of lessthan 100%, (b) contains 3,4-microstructure, (c) contains1,2-microstructure, (d) has a weight average molecular weight of lessthan 100,000, or (e) is free of residual proteins, soaps, lipids,resins, or sugars indicative of natural rubber. In some embodiments, themethod further comprises analyzing the ¹⁴C content of the polymer andverifying that the f_(M) value is greater than 0.9.

In one aspect, provided is a method for verifying that a copolymerhaving repeat units that are derived from isoprene contains isoprenethat is from a sustainable renewable non-petroleum derived source, saidmethod comprising: (I) determining the δ¹³C value of at least onepolyisoprene block in the copolymer; and (II) verifying that theisoprene in the copolymer is from a sustainable renewable non-petroleumderived source if the polyisoprene block has (i) a δ¹³C value of greaterthan −22‰, or (ii) a δ¹³C value which is within the range of −34‰ to−28.5. In some embodiments, the method further comprises analyzing the¹⁴C content of the polymer and verifying that the f_(M) value is greaterthan 0.9.

In some embodiments of any of the aspects, the isoprene monomer isproduced by cells in culture. In some embodiments, the cells in cultureare capable of producing greater than about 400 nmole or about 1000nmole of isoprene monomer/gram of cells for the wet weight of thecells/hour (nmole/g_(wcm)/hr) of isoprene monomer. In some embodiments,the cells have a heterologous nucleic acid that (i) encodes an isoprenesynthase polypeptide and (ii) is operably linked to a promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate, glycerol,glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animaloil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride,diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing. Insome embodiments, the cells are cultured under limited glucoseconditions.

In some embodiments, the cells in culture are capable of converting morethan about 0.002% of the carbon in a cell culture medium into isoprenemonomer. In some embodiments, the cells have a heterologous nucleic acidthat (i) encodes an isoprene synthase polypeptide and (ii) is operablylinked to a promoter. In some embodiments, the cells are cultured in aculture medium that includes a carbon source, such as, but not limitedto, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In some embodiments, the cells in culture comprise a heterologousnucleic acid encoding an isoprene synthase polypeptide. In someembodiments, the cells have a heterologous nucleic acid that (i) encodesan isoprene synthase polypeptide and (ii) is operably linked to apromoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In some embodiments, the cells in culture are capable of producing anamount of isoprene monomer (such as the total amount of isopreneproduced or the amount of isoprene produced per liter of broth per hourper OD₆₀₀) during stationary phase is greater than or about 2 or moretimes the amount of isoprene monomer produced during the growth phasefor the same length of time. In some embodiments, the cells in cultureare capable of producing isoprene monomer only in stationary phase. Insome embodiments, the cells in culture are capable of producing isoprenemonomer in both the growth phase and stationary phase. In variousembodiments, the cells in culture are capable of producing an amount ofisoprene monomer during stationary phase is greater than or about 2, 3,4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene monomerproduced during the growth phase for the same length of time.

In some embodiments of any of the aspects, isoprene of the isoprenemonomer is from a composition. In some embodiments, the compositioncomprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprenemonomer. In some embodiments, the composition comprises greater than orabout 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprenemonomer (w/w) of the volatile organic fraction of the composition isisoprene monomer.

In some embodiments, the composition comprises greater than or about99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene monomer by weightcompared to the total weight of all C5 hydrocarbons in the composition.In some embodiments, the composition comprises less than or about 0.12,0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001,0.00005, or 0.00001% C5 hydrocarbons other than isoprene monomer (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the composition. In some embodiments, the compositionhas less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne byweight compared to the total weight of all C5 hydrocarbons in thecomposition. In particular embodiments, the composition has greater thanabout 2 mg of isoprene monomer and has greater than or about 99.90,99.92, 99.94, 99.96, 99.98, or 100% isoprene monomer by weight comparedto the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the composition has less than or about 50, 40, 30,20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound thatinhibits the polymerization of isoprene monomer for any compound in thecomposition that inhibits the polymerization of isoprene monomer. Inparticular embodiments, the composition also has greater than about 2 mgof isoprene monomer.

In some embodiments, the composition has one or more compounds selectedfrom the group consisting of ethanol, acetone, C5 prenyl alcohols, andisoprenoid compounds with 10 or more carbon atoms. In some embodiments,the composition has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5,1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),or any two or more of the foregoing. In particular embodiments, thecomposition has greater than about 2 mg of isoprene monomer and has oneor more compounds selected from the group consisting of ethanol,acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or morecarbon atoms.

In some embodiments, the composition includes isoprene monomer and oneor more second compounds selected from the group consisting of2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,citronellol and geraniol. In various embodiments, the amount of one ofthese second components relative to the amount of isoprene monomer inunits of percentage by weight (i.e., weight of the component divided bythe weight of isoprene times 100) is at greater than or about 0.01,0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or110% (w/w).

In some embodiments, the composition comprises (i) a gas phase thatcomprises isoprene monomer and (ii) cells in culture that producegreater than about 400 nmole/g_(wcm)/hr of isoprene. In someembodiments, the composition comprises a closed system, and the gasphase comprises greater than or about 5. 10, 20, 30, 40, 50, 60, 70, 80,90, 100 μg/L of isoprene monomer when normalized to 1 mL of 1 OD₆₀₀cultured for 1 hour. In some embodiments, the composition comprises anopen system, and the gas phase comprises greater than or about 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100 μg/L of isoprene monomer whensparged at a rate of 1 vvm. In some embodiments, the volatile organicfraction of the gas phase comprises greater than or about 99.90, 99.92,99.94, 99.96, 99.98, or 100% isoprene monomer by weight compared to thetotal weight of all C5 hydrocarbons in the volatile organic fraction. Insome embodiments, the volatile organic fraction of the gas phasecomprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01,0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons otherthan isoprene monomer (such 1,3-cyclopentadiene, cis-1,3-pentadiene,trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene,2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared tothe total weight of all C5 hydrocarbons in the volatile organicfraction. In some embodiments, the volatile organic fraction of the gasphase has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01,0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne by weight compared to the total weight of all C5hydrocarbons in the volatile organic fraction. In particularembodiments, the volatile organic fraction of the gas phase has greaterthan about 2 mg of isoprene monomer and has greater than or about 99.90,99.92, 99.94, 99.96, 99.98, or 100% isoprene monomer by weight comparedto the total weight of all C5 hydrocarbons in the volatile organicfraction.

In some embodiments, the volatile organic fraction of the gas phase ofthe composition has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5,0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits thepolymerization of isoprene for any compound in the volatile organicfraction of the gas phase that inhibits the polymerization of isoprenemonomer. In particular embodiments, the volatile organic fraction of thegas phase also has greater than about 2 mg of isoprene monomer.

In some embodiments, the volatile organic fraction of the gas phase ofthe composition has one or more compounds selected from the groupconsisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoidcompounds with 10 or more carbon atoms. In some embodiments, thevolatile organic fraction of the gas phase has greater than or about0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120μg/L of ethanol, acetone, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more ofthe foregoing. In particular embodiments, the volatile organic fractionof the gas phase has greater than about 2 mg of isoprene monomer and hasone or more compounds selected from the group consisting of ethanol,acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or morecarbon atoms.

In some embodiments, the volatile organic fraction of the gas phase ofthe composition has includes isoprene monomer and one or more secondcompounds selected from the group consisting of 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,citronellol and geraniol. In various embodiments, the amount of one ofthese second components relative to amount of isoprene monomer in unitsof percentage by weight (i.e., weight of the component divided by theweight of isoprene times 100) is at greater than or about 0.01, 0.02,0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%(w/w) in the volatile organic fraction of the gas phase.

In some embodiments of any of the compositions, at least a portion ofthe isoprene monomer is in a gas phase. In some embodiments, at least aportion of the isoprene monomer is in a liquid phase (such as acondensate). In some embodiments, at least a portion of the isoprenemonomer is in a solid phase. In some embodiments, at least a portion ofthe isoprene monomer is adsorbed to a solid support, such as a supportthat includes silica and/or activated carbon. In some embodiments, thecomposition includes ethanol. In some embodiments, the compositionincludes between about 75 to about 90% by weight of ethanol, such asbetween about 75 to about 80%, about 80 to about 85%, or about 85 toabout 90% by weight of ethanol. In some embodiments, the compositionincludes between about 4 to about 15% by weight of isoprene monomer,such as between about 4 to about 8%, about 8 to about 12%, or about 12to about 15% by weight of isoprene monomer.

In some embodiments, the cells in culture are from a system thatincludes a reactor chamber wherein the cells are capable of producinggreater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hrisoprene monomer. In some embodiments, the system is not a closedsystem. In some embodiments, at least a portion of the isoprene monomeris removed from the system. In some embodiments, the system includes agas phase comprising isoprene monomer. In various embodiments, the gasphase comprises any of the compositions described herein.

In one aspect is provided a tire comprising any polyisoprene polymerdescribed herein. For example, in one embodiment is provided tirecomprising a polyisoprene polymer which is comprised of repeat unitsthat are derived from isoprene monomer, wherein the polyisoprene polymerhas δ¹³C value of greater than −22‰, or a δ¹³C value which is within therange of −30‰ to −28.5‰, −32‰ to −24‰, or −34‰ to −24‰. In some of theseembodiments, the polyisoprene is free of protein. In some embodiments,the polyisoprene is a polyisoprene homopolymer.

In some embodiments, the polyisoprene polymer described herein isproduced by (i) polymerizing isoprene monomer in any of the compositionsdescribed herein or (ii) polymerizing isoprene recovered from any of thecompositions described herein. In some embodiments are provided methodsof producing any polyisoprene polymer described herein by (i)polymerizing isoprene monomer in any of the compositions describedherein or (ii) polymerizing isoprene recovered from any of thecompositions described herein.

In some embodiments of any of the compositions, systems, and methodsdescribed herein, a nonflammable concentration of isoprene monomer inthe gas phase is produced. In some embodiments, the gas phase comprisesless than about 9.5% (volume) oxygen. In some embodiments, the gas phasecomprises greater than or about 9.5% (volume) oxygen, and theconcentration of isoprene monomer in the gas phase is less than thelower flammability limit or greater than the upper flammability limit.In some embodiments, the portion of the gas phase other than isoprenemonomer comprises between about 0% to about 100% (volume) oxygen, suchas between about 10% to about 100% (volume) oxygen. In some embodiments,the portion of the gas phase other than isoprene monomer comprisesbetween about 0% to about 99% (volume) nitrogen. In some embodiments,the portion of the gas phase other than isoprene monomer comprisesbetween about 1% to about 50% (volume) CO₂.

In some embodiments, the cells in culture produce isoprene monomer atgreater than or about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hrisoprene. In some embodiments, the cells in culture convert greater thanor about 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6%, or more of thecarbon in the cell culture medium into isoprene monomer. In someembodiments, the cells in culture produce isoprene monomer at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 100,000, or more ng of isoprene monomer/gram of cells forthe wet weight of the cells/hr (ng/g_(wcm)/h). In some embodiments, thecells in culture produce a cumulative titer (total amount) of isoprenemonomer at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300,400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500,3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/Lof broth (mg/L_(broth), wherein the volume of broth includes the volumeof the cells and the cell medium). Other exemplary rates of isoprenemonomer production and total amounts of isoprene monomer production aredisclosed herein.

In some embodiments of any of the aspects, the cells in culture furthercomprise a heterologous nucleic acid encoding an IDI polypeptide. Insome embodiments, the cells further comprise an insertion of a copy ofan endogenous nucleic acid encoding an IDI polypeptide. In someembodiments, the cells further comprise a heterologous nucleic acidencoding a DXS polypeptide. In some embodiments, the cells furthercomprise an insertion of a copy of an endogenous nucleic acid encoding aDXS polypeptide. In some embodiments, the cells further comprise one ormore nucleic acids encoding an IDI polypeptide and a DXS polypeptide. Insome embodiments, one nucleic acid encodes the isoprene synthasepolypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments,one vector encodes the isoprene synthase polypeptide, IDI polypeptide,and DXS polypeptide. In some embodiments, the vector comprises aselective marker, such as an antibiotic resistance nucleic acid.

In some embodiments, the heterologous isoprene synthase nucleic acid isoperably linked to a T7 promoter, such as a T7 promoter contained in amedium or high copy plasmid. In some embodiments, the heterologousisoprene synthase nucleic acid is operably linked to a Trc promoter,such as a Trc promoter contained in a medium or high copy plasmid. Insome embodiments, the heterologous isoprene synthase nucleic acid isoperably linked to a Lac promoter, such as a Lac promoter contained in alow copy plasmid. In some embodiments, the heterologous isoprenesynthase nucleic acid is operably linked to an endogenous promoter, suchas an endogenous alkaline serine protease promoter. In some embodiments,the heterologous isoprene synthase nucleic acid integrates into achromosome of the cells without a selective marker.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

In some embodiments, at least a portion of the cells in culture maintainthe heterologous isoprene synthase nucleic acid for at least or about 5,10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture(such as a continuous culture without dilution). In some embodiments,the nucleic acid comprising the isoprene synthase, IDI, or DXS nucleicacid also comprises a selective marker, such as an antibiotic resistancenucleic acid.

In some embodiments, the cells in culture further comprise aheterologous nucleic acid encoding an MVA pathway polypeptide (such asan MVA pathway polypeptide from Saccharomyces cerevisia or Enterococcusfaecalis). In some embodiments, the cells further comprise an insertionof a copy of an endogenous nucleic acid encoding an MVA pathwaypolypeptide (such as an MVA pathway polypeptide from Saccharomycescerevisia or Enterococcus faecalis). In some embodiments, the cellscomprise an isoprene synthase, DXS, and MVA pathway nucleic acid. Insome embodiments, the cells comprise an isoprene synthase nucleic acid,a DXS nucleic acid, an IDI nucleic acid, and a MVA pathway nucleic (inaddition to the IDI nucleic acid).

In some embodiments, the isoprene synthase polypeptide is anaturally-occurring polypeptide from a plant such as Pueraria (e.g.,Pueraria montana or Pueraria lobata).

In some embodiments, the cells in culture are bacterial cells, such asgram-positive bacterial cells (e.g., Bacillus cells such as Bacillussubtilis cells or Streptomyces cells such as Streptomyces lividans,Streptomyces coelicolor, or Streptomyces griseus cells). In someembodiments, the cells in culture are gram-negative bacterial cells(e.g., Escherichia cells such as Escherichia coli cells or Pantoea cellssuch as Pantoea citrea cells). In some embodiments, the cells in cultureare fungal, cells such as filamentous fungal cells (e.g., Trichodermacells such as Trichoderma reesei cells or Aspergillus cells such asAspergillus oryzae and Aspergillus niger) or yeast cells (e.g., Yarrowiacells such as Yarrowia lipolytica cells).

In some embodiments, the microbial polypeptide carbon source includesone or more polypeptides from yeast or bacteria. In some embodiments,the plant polypeptide carbon source includes one or more polypeptidesfrom soy, corn, canola, jatropha, palm, peanut, sunflower, coconut,mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, orlinseed.

In one aspect, the invention features a product produced by any of thecompositions or methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of a kudzu isoprene synthase genecodon-optimized for expression in E. coli (SEQ ID NO:1). The atg startcodon is in italics, the stop codon is in bold and the added PstI siteis underlined.

FIG. 2 is a map of pTrcKudzu.

FIG. 3A depicts the first portion of the nucleotide sequence ofpTrcKudzu (SEQ ID NO:2). The RBS is underlined, the kudzu isoprenesynthase start codon is in bold capitol letters and the stop codon is inbold, capitol, italics letters. The vector backbone is pTrcHis2B.

FIG. 3B depicts an intermediate portion of the nucleotide sequence ofthe pTrcKudzu which follows the sequence shown in FIG. 3A.

FIG. 3C depicts the final portion of the nucleotide sequence of thepTrcKudzu which follows the sequence shown in FIG. 3B.

FIG. 4 is a map of pETNHisKudzu.

FIG. 5A depicts the first portion of the nucleotide sequence ofpETNHisKudzu (SEQ ID NO:5).

FIG. 5B depicts an intermediate portion of the nucleotide sequence ofpETNHisKudzu which follows the sequence shown in FIG. 5A.

FIG. 5C depicts the final portion of the nucleotide sequence ofpETNHisKudzu which follows the sequence shown in FIG. 5B.

FIG. 6 is a map of pCL-lac-Kudzu.

FIG. 7A depicts the first portion of the nucleotide sequence ofpCL-lac-Kudzu (SEQ ID NO:7).

FIG. 7B depicts an intermediate portion of the nucleotide sequence ofpCL-lac-Kudzu which follows the sequence shown in FIG. 7A.

FIG. 7C depicts the final portion of the nucleotide sequence ofpCL-lac-Kudzu which follows the sequence shown in FIG. 7B.

FIG. 8A is a graph showing the production of isoprene in E. coli BL21cells with no vector.

FIG. 8B is a graph showing the production of isoprene in E. coli BL21cells with pCL-lac-Kudzu

FIG. 8C is a graph showing the production of isoprene in E. coli BL21cells with pTrcKudzu.

FIG. 8D is a graph showing the production of isoprene in E. coli BL21cells with pETN-HisKudzu.

FIG. 9A is a graph showing OD over time of fermentation of E. coliBL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 9B is a graph showing isoprene production over time of fermentationof E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 10A is a graph showing the production of isoprene in Panteoacitrea. Control cells without recombinant kudzu isoprene synthase. Greydiamonds represent isoprene synthesis, black squares represent OD₆₀₀.

FIG. 10B is a graph showing the production of isoprene in Panteoa citreaexpressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis,black squares represent OD₆₀₀.

FIG. 10C is a graph showing the production of isoprene in Panteoa citreaexpressing pTrcKudzu. Grey diamonds represent isoprene synthesis, blacksquares represent OD₆₀₀.

FIG. 11 is a graph showing the production of isoprene in Bacillussubtilis expressing recombinant isoprene synthase. BG3594comK is a B.subtilis strain without plasmid (native isoprene production).CF443-BG3594comK is a B. subtilis strain with pBSKudzu (recombinantisoprene production). IS on the y-axis indicates isoprene.

FIG. 12A depicts the first portion of the nucleotide sequence of pBSKudzu #2 (SEQ ID NO:57).

FIG. 12B depicts an intermediate portion of the nucleotide sequence ofpBS Kudzu #2 which follows the sequence shown in FIG. 12A.

FIG. 12C depicts the final portion of the nucleotide sequence of pBSKudzu #2 which follows the sequence shown in FIG. 12B.

FIG. 13 is the nucleotide sequence of kudzu isoprene synthasecodon-optimized for expression in Yarrowia (SEQ ID NO:8).

FIG. 14 is a map of pTrex3g comprising a kudzu isoprene synthase genecodon-optimized for expression in Yarrowia.

FIG. 15A depicts the first portion of the nucleotide sequence of vectorpSPZ1(MAP29spb) (SEQ ID NO:11).

FIG. 15B depicts an intermediate portion of the nucleotide sequence ofvector pSPZ1(MAP29spb) which follows the sequence shown in FIG. 15A.

FIG. 15C depicts the final portion of the nucleotide sequence of vectorpSPZ1(MAP29spb) which follows the sequence shown in FIG. 15B.

FIG. 16 is the nucleotide sequence of the synthetic kudzu (Puerariamontana) isoprene gene codon-optimized for expression in Yarrowia (SEQID NO:12).

FIG. 17 is the nucleotide sequence of the synthetic hybrid poplar(Populus alba x Populus tremula) isoprene synthase gene (SEQ ID NO:13).The ATG start codon is in bold and the stop codon is underlined.

FIG. 18A (FIGS. 18A1 and 18A2) shows a schematic outlining constructionof vectors pYLA 1, pYL1 and pYL2 (SEQ ID NOS: 73, 74, 75, 76, 77 and79).

FIG. 18B shows a schematic outlining construction of the vectorpYLA(POP1) (SEQ ID NOS: 71 and 72).

FIG. 18C shows a schematic outlining construction of the vectorpYLA(KZ1).

FIG. 18D shows a schematic outlining construction of the vectorpYLI(KZ1) (SEQ ID NOS: 69 and 70).

FIG. 18E shows a schematic outlining construction of the vectorpYLI(MAP29).

FIG. 18F shows a schematic outlining construction of the vectorpYLA(MAP29).

FIG. 19A shows the MVA and DXP metabolic pathways for isoprene (based onF. Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). Thefollowing description includes alternative names for each polypeptide inthe pathways and a reference that discloses an assay for measuring theactivity of the indicated polypeptide (each of these references are eachhereby incorporated by reference in their entireties, particularly withrespect to assays for polypeptide activity for polypeptides in the MVAand DXP pathways). Mevalonate Pathway: AACT; Acetyl-CoAacetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184:2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC2.3.3.10. Assay: J. Bacteriol., 184: 4065-4070, 2002; HMGR;3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay: J.Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC2.7.1.36. Assay: Curr Genet. 19:9-14, 1991. PMK; Phosphomevalonatekinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol., 11:620-631, 1991;DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC 4.1.1.33. Assay:Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphatedelta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem.264:19169-19175, 1989. DXP Pathway: DXS; 1-Deoxyxylulose-5-phosphatesynthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR;1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7. Assay:Eur. J. Biochem. 269:4446-4457, 2002; MCT;4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60.Assay: PNAS, 97: 6451-6456, 2000; CMK;4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148.Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay: PNAS,96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. Org. Chem.,70:9168-9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphatereductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.

FIG. 19B illustrates the classical and modified MVA pathways. 1,acetyl-CoA acetyltransferase (AACT); 2, HMG-CoA synthase (HMGS); 3,HMG-CoA reductase (HMGR); 4, mevalonate kinase (MVK); 5,phosphomevalonate kinase (PMK); 6, diphosphomevalonate decarboxylase(MVD or DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8,phosphomevalonate decarboxylase (PMDC); 9, isopentenyl phosphate kinase(IPK). The classical MVA pathway proceeds from reaction 1 throughreaction 7 via reactions 5 and 6, while a modified MVA pathway goesthrough reactions 8 and 9. P and PP in the structural formula arephosphate and pyrophosphate, respectively. This figure was taken fromKoga and Morii, Microbiology and Mol. Biology Reviews, 71:97-120, 2007,which is incorporated by reference in its entirety, particular withrespect to nucleic acids and polypeptides of the modified MVA pathway.The modified MVA pathway is present, for example, in some archaealorganisms, such as Methanosarcina mazei.

FIG. 20A is a graph representing results of the GC-MS analysis ofisoprene production by recombinant Y. lipolytica strains without (left)or with (right) a kudzu isoprene synthase gene. The arrows indicate theelution time of the authentic isoprene standard.

FIG. 20 B is a graph representing results of the GC-MS analysis ofisoprene production by recombinant Y. lipolytica strains with a kudzuisoprene synthase gene. The arrows indicate the elution time of theauthentic isoprene standard.

FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.

FIG. 22A depicts the first portion of the nucleotide sequence ofpTrcKudzu yIDI DXS Kan (SEQ ID NO:20).

FIG. 22B depicts an intermediate portion of the nucleotide sequence ofpTrcKudzu yIDI DXS Kan which follows the sequence shown in FIG. 22A.

FIG. 22C depicts an intermediate portion of the nucleotide sequence ofpTrcKudzu yIDI DXS Kan which follows the sequence shown in FIG. 22B.

FIG. 22D depicts the final portion of the nucleotide sequence ofpTrcKudzu yIDI DXS Kan which follows the sequence shown in FIG. 22C.

FIG. 23A is a graph showing production of isoprene from glucose inBL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 μmol).The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axisis total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23B is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23C is a graph showing production of isoprene from glucose inBL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23D is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG(400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ andthe y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀,circles represent total isoprene productivity (μg/L) and squaresrepresent specific productivity of isoprene (μg/L/OD).

FIG. 23E is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23F is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23G is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23H is a graph showing production of isoprene from glucose inBL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction withIPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀and the y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Black diamonds representOD₆₀₀, black triangles represent isoprene productivity (μg/L) and whitesquares represent specific productivity of isoprene (μg/L/OD).

FIG. 24 is a map of pTrcKKDyIkIS kan.

FIG. 25A depicts the first portion of the nucleotide sequence ofpTrcKKDyIkIS kan (SEQ ID NO:33).

FIG. 25B depicts an intermediate portion of the nucleotide sequence ofpTrcKKDyIkIS kan which follows the sequence shown in FIG. 25A.

FIG. 25C depicts an intermediate portion of the nucleotide sequence ofpTrcKKDyIkIS kan which follows the sequence shown in FIG. 25B.

FIG. 25D depicts the final portion of the nucleotide sequence ofpTrcKKDyIkIS kan which follows the sequence shown in FIG. 25C.

FIG. 26 is a map of pCL PtrcUpperPathway.

FIG. 27A depicts the first portion of the nucleotide sequence of pCLPtrcUpperPathway (SEQ ID NO:46).

FIG. 27B depicts an intermediate portion of the nucleotide sequence ofpCL PtrcUpperPathway which follows the sequence shown in FIG. 27A.

FIG. 27C depicts an intermediate portion of the nucleotide sequence ofpCL PtrcUpperPathway which follows the sequence shown in FIG. 27B.

FIG. 27D depicts the final portion of the nucleotide sequence of pCLPtrcUpperPathway which follows the sequence shown in FIG. 27C.

FIG. 28 shows a map of the cassette containing the lower MVA pathway andyeast idi for integration into the B. subtilis chromosome at the nprElocus. nprE upstream/downstream indicates 1 kb each of sequence from thenprE locus for integration. aprE promoter (alkaline serine proteasepromoter) indicates the promoter (−35, −10, +1 transcription start site,RBS) of the aprE gene. MVK1 indicates the yeast mevalonate kinase gene.RBS-PMK indicates the yeast phosphomevalonte kinase gene with a BacillusRBS upstream of the start site. RBS-MPD indicates the yeastdiphosphomevalonate decarboxylase gene with a Bacillus RBS upstream ofthe start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBSupstream of the start site. Terminator indicates the terminator alkalineserine protease transcription terminator from B. amyliquefaciens. SpecRindicates the spectinomycin resistance marker. “nprE upstream repeat foramp.” indicates a direct repeat of the upstream region used foramplification.

FIG. 29A depicts the first portion of the nucleotide sequence ofcassette containing the lower MVA pathway and yeast idi for integrationinto the B. subtilis chromosome at the nprE locus (SEQ ID NO:47).

FIG. 29B depicts an intermediate portion of the nucleotide sequence ofcassette containing the lower MVA pathway and yeast idi for integrationinto the B. subtilis chromosome at the nprE locus which follows thesequence shown in FIG. 29A.

FIG. 29C depicts an intermediate portion of the nucleotide sequence ofcassette containing the lower MVA pathway and yeast idi for integrationinto the B. subtilis chromosome at the nprE locus which follows thesequence shown in FIG. 29B.

FIG. 29D depicts the final portion of the nucleotide sequence ofcassette containing the lower MVA pathway and yeast idi for integrationinto the B. subtilis chromosome at the nprE locus which follows thesequence shown in FIG. 29C.

FIG. 30 is a map of p9796-poplar.

FIG. 31A depicts the first portion of the nucleotide sequence ofp9796-poplar (SEQ ID NO:48).

FIG. 31B depicts the final portion of the nucleotide sequence ofp9796-poplar which follows the sequence shown in FIG. 31A.

FIG. 32 is a map of pTrcPoplar.

FIG. 33A depicts the first portion of the nucleotide sequence ofpTrcPoplar (SEQ ID NO:49).

FIG. 33B depicts an intermediate portion of the nucleotide sequence ofpTrcPoplar which follows the sequence shown in FIG. 33A.

FIG. 33C depicts the final portion of the nucleotide sequence ofpTrcPoplar which follows the sequence shown in FIG. 33B.

FIG. 34 is a map of pTrcKudzu yIDI Kan.

FIG. 35A depicts the first portion of the nucleotide sequence ofpTrcKudzu yIDI Kan (SEQ ID NO:50).

FIG. 35B depicts an intermediate portion of the nucleotide sequence ofpTrcKudzu yIDI Kan which follows the sequence shown in FIG. 35A.

FIG. 35C depicts the final portion of the nucleotide sequence ofpTrcKudzu yIDI Kan which follows the sequence shown in FIG. 35B.

FIG. 36 is a map of pTrcKudzuDXS Kan.

FIG. 37A depicts the first portion of the nucleotide sequence ofpTrcKudzuDXS Kan (SEQ ID NO:51).

FIG. 37B depicts an intermediate portion of the nucleotide sequence ofpTrcKudzuDXS Kan which follows the sequence shown in FIG. 37A.

FIG. 37C depicts the final portion of the nucleotide sequence ofpTrcKudzuDXS Kan which follows the sequence shown in FIG. 37B.

FIG. 38 is a map of pCL PtrcKudzu.

FIG. 39A depicts the first portion of the nucleotide sequence of pCLPtrcKudzu (SEQ ID NO:52).

FIG. 39B depicts an intermediate portion of the nucleotide sequence ofpCL PtrcKudzub which follows the sequence shown in FIG. 39A.

FIG. 39C depicts the final portion of the nucleotide sequence of pCLPtrcKudzub which follows the sequence shown in FIG. 39B.

FIG. 40 is a map of pCL PtrcKudzu A3.

FIG. 41A depicts the first portion of the nucleotide sequence of pCLPtrcKudzu A3 (SEQ ID NO:53).

FIG. 41B depicts an intermediate portion of the nucleotide sequence ofpCL PtrcKudzu A3 which follows the sequence shown in FIG. 41A.

FIG. 41C depicts the final portion of the nucleotide sequence of pCLPtrcKudzu A3 which follows the sequence shown in FIG. 41B.

FIG. 42 is a map of pCL PtrcKudzu yIDI.

FIG. 43A depicts the first portion of the nucleotide sequence of pCLPtrcKudzu yIDI (SEQ ID NO:54).

FIG. 43B depicts an intermediate portion of the nucleotide sequence ofpCL PtrcKudzu yIDI which follows the sequence shown in FIG. 43A.

FIG. 43C depicts the final portion of the nucleotide sequence of pCLPtrcKudzu yIDI which follow the sequence shown in FIG. 43B.

FIG. 44 is a map of pCL PtrcKudzu DXS.

FIG. 45A depicts the first portion of the nucleotide sequence of pCLPtrcKudzu DXS (SEQ ID NO:55).

FIG. 45B depicts an intermediate portion of the nucleotide sequence ofpCL PtrcKudzu DXS which follows the sequence shown in FIG. 45A.

FIG. 45C depicts an intermediate portion of the nucleotide sequence ofpCL PtrcKudzu DXS which follows the sequence shown in FIG. 45B.

FIG. 45D depicts the final portion of the nucleotide sequence of pCLPtrcKudzu DXS which follows the sequence shown in FIG. 45C.

FIG. 46A is a graph representing isoprene production from corn stoverfeedstock at a function of time. Grey squares represent OD₆₀₀measurements of the cultures at the indicated times post-inoculation andblack triangles represent isoprene production at the indicated timespost-inoculation.

FIG. 46B is a graph representing isoprene production from bagassefeedstocks. Grey squares represent OD₆₀₀ measurements of the cultures atthe indicated times post-inoculation and black triangles representisoprene production at the indicated times post-inoculation.

FIG. 46C is a graph representing isoprene production from softwood stockfeedstock Grey squares represent OD₆₀₀ measurements of the cultures atthe indicated times post-inoculation and black triangles representisoprene production at the indicated times post-inoculation.

FIG. 46D is a graph representing isoprene production from glucosefeedstock. Grey squares represent OD₆₀₀ measurements of the cultures atthe indicated times post-inoculation and black triangles representisoprene production at the indicated times post-inoculation.

FIG. 46E is a graph representing isoprene production from cells with noadditional feedstock. Grey squares represent OD₆₀₀ measurements of thecultures at the indicated times post-inoculation and black trianglesrepresent isoprene production at the indicated times post-inoculation.

FIG. 47A shows a graph representing isoprene production by BL21 (λDE3)pTrcKudzu yIDI DXS (kan) in a culture with no glucose added. Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47B shows a graph representing isoprene production from 1% glucosefeedstock invert sugar by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47C shows a graph representing isoprene production from 1% invertsugar feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47D shows a graph representing isoprene production from 1% AFEXcorn stover feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 48 shows graphs demonstrating the effect of yeast extract ofisoprene production. Panel A shows the time course of optical densitywithin fermentors fed with varying amounts of yeast extract. Panel Bshows the time course of isoprene titer within fermentors fed withvarying amounts of yeast extract. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. Panel C shows theeffect of yeast extract on isoprene production in E. coli grown infed-batch culture.

FIG. 49 shows graphs demonstrating isoprene production from a 500 Lbioreactor with E. coli cells containing the pTrcKudzu+yIDI+DXS plasmid.Panel A shows the time course of optical density within the 500-Lbioreactor fed with glucose and yeast extract. Panel B shows the timecourse of isoprene titer within the 500-L bioreactor fed with glucoseand yeast extract. The titer is defined as the amount of isopreneproduced per liter of fermentation broth. Panel C shows the time courseof total isoprene produced from the 500-L bioreactor fed with glucoseand yeast extract.

FIG. 50 is a map of pJMupperpathway2.

FIG. 51A depicts the first portion of the nucleotide sequence ofpJMupperpathway2 (SEQ ID NO:56).

FIG. 51B depicts an intermediate portion of the nucleotide sequence ofpJMupperpathway2 which follows the sequence shown in FIG. 51A.

FIG. 51C depicts the final portion of the nucleotide sequence ofpJMupperpathway2 which follows the sequence shown in FIG. 51B.

FIG. 52 is a map of pBS Kudzu #2.

FIG. 53A is a graph showing growth during fermentation time of Bacillusexpressing recombinant kudzu isoprene synthase in 14 liter fed batchfermentation. Black diamonds represent a control strain (BG3594comK)without recombinant isoprene synthase (native isoprene production) andgrey triangles represent Bacillus with pBSKudzu (recombinant isopreneproduction).

FIG. 53B is a graph showing isoprene production during fermentation timeof Bacillus expressing recombinant kudzu isoprene synthase in 14 literfed batch fermentation. Black diamonds represent a control strain(BG3594comK) without recombinant isoprene synthase (native isopreneproduction) and grey triangles represent Bacillus with pBSKudzu(recombinant isoprene production).

FIG. 54 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 55 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 56 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 57 is a time course of optical density within the 15-L bioreactorfed with glycerol.

FIG. 58 is a time course of isoprene titer within the 15-L bioreactorfed with glycerol. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 59 is a time course of total isoprene produced from the 15-Lbioreactor fed with glycerol.

FIGS. 60A-60C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 150-L bioreactor fed withglucose.

FIGS. 61A-61C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 15-L bioreactor fed withglucose.

FIGS. 62A-62C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 15-L bioreactor fed withglucose.

FIG. 63A-63C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 64A-64C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 65A-65C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 66A-66C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 67A-67C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 68 is a graph of the calculated adiabatic flame temperatures forSeries A as a function of fuel concentration for various oxygen levels.The figure legend lists the curves in the order in which they appear inthe graph. For example, the first entry in the figure legend (isoprenein air at 40° C.) corresponds to the highest curve in the graph.

FIG. 69 is a graph of the calculated adiabatic flame temperatures forSeries B as a function of fuel concentration for various oxygen levelswith 4% water. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 70 is a graph of the calculated adiabatic flame temperatures forSeries C as a function of fuel concentration for various oxygen levelswith 5% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 71 is a graph of the calculated adiabatic flame temperatures forSeries D as a function of fuel concentration for various oxygen levelswith 10% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 72 is a graph of the calculated adiabatic flame temperatures forSeries E as a function of fuel concentration for various oxygen levelswith 15% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 73 is a graph of the calculated adiabatic flame temperatures forSeries F as a function of fuel concentration for various oxygen levelswith 20% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 74 is a graph of the calculated adiabatic flame temperatures forSeries G as a function of fuel concentration for various oxygen levelswith 30% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 75A is a table of the conversion of the CAFT Model results fromweight percent to volume percent for series A.

FIG. 75B is a graph of the flammability results from the CAFT model forSeries A in FIG. 68 plotted as volume percent.

FIG. 76A is a table of the conversion of the CAFT Model results fromweight percent to volume percent for series B.

FIG. 76B is a graph of the flammability results from the CAFT model forSeries B in FIG. 69 plotted as volume percent.

FIG. 77 is a figure of the flammability test vessel.

FIG. 78A is a graph of the flammability Curve for Test Series 1: 0%Steam, 0 psig, and 40° C.

FIG. 78B is a table summarizing the explosion and non-explosion datapoints for Test Series 1.

FIG. 78C is a graph of the flammability curve for Test Series 1 comparedwith the CAFT Model.

FIG. 79A is a graph of the flammability curve for Test Series 2: 4%Steam, 0 psig, and 40° C.

FIG. 79B is a table summarizing the explosion and non-explosion datapoints for Test Series 2.

FIG. 79C is a graph of the flammability curve for Test Series 2 comparedwith the CAFT Model.

FIGS. 80A and 80B are a table of the detailed experimental conditionsand results for Test Series 1.

FIG. 81 is a table of the detailed experimental conditions and resultsfor Test Series 2.

FIG. 82 is a graph of the calculated adiabatic flame temperature plottedas a function of fuel concentration for various nitrogen/oxygen ratiosat 3 atmospheres of pressure.

FIG. 83 is a graph of the calculated adiabatic flame temperature plottedas a function of fuel concentration for various nitrogen/oxygen ratiosat 1 atmosphere of pressure.

FIG. 84 is a graph of the flammability envelope constructed using datafrom FIG. 82 and following the methodology described in Example 13. Theexperimental data points (circles) are from tests described herein thatwere conducted at 1 atmosphere initial system pressure.

FIG. 85 is a graph of the flammability envelope constructed using datafrom FIG. 83 and following the methodology described in Example 13. Theexperimental data points (circles) are from tests described herein thatwere conducted at 1 atmosphere initial system pressure.

FIG. 86A is a GC/MS chromatogram of fermentation off-gas.

FIG. 86B is an expansion of FIG. 86A to show minor volatiles present infermentation off-gas.

FIG. 87A is a GC/MS chromatogram of trace volatiles present in off-gasfollowing cryo-trapping at −78° C.

FIG. 87B is a GC/MS chromatogram of trace volatiles present in off-gasfollowing cryo-trapping at −196° C.

FIG. 87C is an expansion of FIG. 87B.

FIG. 87D is an expansion of FIG. 87C.

FIGS. 88A and 88B are GC/MS chromatogram comparing C₅ hydrocarbons frompetroleum-derived isoprene (FIG. 88A) and biologically produced isoprene(FIG. 88B). The standard contains three C₅ hydrocarbon impuritieseluting around the main isoprene peak (FIG. 88A). In contrast,biologically produced isoprene contains amounts of ethanol and acetone(run time of 3.41 minutes) (FIG. 88A).

FIG. 89 is a graph of the analysis of fermentation off-gas of an E. coliBL21 (DE3) pTrcIS strain expressing a Kudzu isoprene synthase and fedglucose with 3 g/L yeast extract.

FIG. 90 shows the structures of several impurities that are structurallysimilar to isoprene and may also act as polymerization catalyst poisons.

FIG. 91 is a map of pTrcHis2AUpperPathway (also called pTrcUpperMVA).

FIGS. 92A-92C are the nucleotide sequence of pTrcHis2AUpperPathway (alsocalled pTrcUpperMVA) (SEQ ID NO:86).

FIG. 93 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 94 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 95 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 96 is a time course of optical density within the 15-L bioreactorfed with invert sugar.

FIG. 97 is a time course of isoprene titer within the 15-L bioreactorfed with invert sugar. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 98 is a time course of total isoprene produced from the 15-Lbioreactor fed with invert sugar.

FIG. 99 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 100 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 101 is a time course of isoprene specific activity from the 15-Lbioreactor fed with glucose.

FIG. 102 is a map of pCLPtrcUpperPathwayHGS2.

FIGS. 103A-103C are the nucleotide sequence of pCLPtrcUpperPathwayHGS2(SEQ ID NO:87).

FIG. 104 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 105 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 106 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 107 is a map of plasmid MCM330.

FIGS. 108A-108C are the nucleotide sequence of plasmid MCM330 (SEQ IDNO:90).

FIG. 109 is a map of pET24D-Kudzu.

FIGS. 110A and 110B are the nucleotide sequence of pET24D-Kudzu (SEQ IDNO:101).

FIG. 111A is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 111B is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 111C is a time course of specific productivity of isoprene in the15-L bioreactor fed with glucose.

FIG. 112 is a map of plasmid pET24 P. alba HGS.

FIG. 113A depicts the first portion of the nucleotide sequence ofplasmid pET24 P. alba HGS (SEQ ID NO:102).

FIG. 113B depicts the final portion of the nucleotide sequence ofplasmid pET24 P. alba HGS which follows the sequence shown in FIG. 113A.

FIG. 114 is a schematic diagram showing restriction sites used forendonuclease digestion to construct plasmid EWL230 and compatiblecohesive ends between BspHI and NcoI sites.

FIG. 115 is a map of plasmid EWL230.

FIG. 116A depicts the first portion of the nucleotide sequence ofplasmid EWL230 (SEQ ID NO:103).

FIG. 116B depicts the final portion of the nucleotide sequence ofplasmid EWL230 which follows the sequence shown in FIG. 116A.

FIG. 117 is a schematic diagram showing restriction sites used forendonuclease digestion to construct plasmid EWL244 and compatiblecohesive ends between NsiI and PstI sites.

FIG. 118 is a map of EWL244.

FIG. 119A depicts the first portion of the nucleotide sequence ofplasmid EWL244 (SEQ ID NO:104).

FIG. 119B depicts the final portion of the nucleotide sequence ofplasmid EWL244 which follows the sequence shown in FIG. 119A.

FIG. 120 is a map of plasmids MCM484-487.

FIG. 121A depicts the first portion of the nucleotide sequence ofplasmid MCM484 (SEQ ID NO:105).

FIG. 121B depicts an intermediate portion of the nucleotide sequence ofplasmid MCM484 following the sequence shown in FIG. 121A.

FIG. 121C depicts the final portion of the nucleotide sequence ofplasmid MCM484 following the sequence shown in FIG. 121AB.

FIG. 122A depicts the first portion of the nucleotide sequence ofplasmid MCM485 (SEQ ID NO:106).

FIG. 122B depicts an intermediate portion of the nucleotide sequence ofplasmid MCM485 which follows the sequence shown in FIG. 122A.

FIG. 122C depicts the final portion of the nucleotide sequence ofplasmid MCM485 which follows the sequence shown in FIG. 122B.

FIG. 123A depicts the first portion of the nucleotide sequence ofplasmid MCM486 (SEQ ID NO:107).

FIG. 123B depicts an intermediate portion of the nucleotide sequence ofplasmid MCM486 which follows the sequence shown in FIG. 123A.

FIG. 123C depicts the final portion of the nucleotide sequence ofplasmid MCM486 which follows the sequence shown in FIG. 123B.

FIG. 124A depicts the first portion of the nucleotide sequence ofplasmid MCM487 (SEQ ID NO:108).

FIG. 124B depicts an intermediate portion of the nucleotide sequence ofplasmid MCM487 which follows the sequence shown in FIG. 124A.

FIG. 124C depicts the final portion of the nucleotide sequence ofplasmid MCM487 which follows the sequence shown in FIG. 124B.

FIGS. 125A-125D are graphs of isoprene production by E. coli strain(EWL256) expressing genes from the MVA pathway and grown in fed-batchculture at the 15-L scale without yeast extract feeding. FIG. 125A showsthe time course of optical density within the 15-L bioreactor fed withglucose. FIG. 125B shows the time course of isoprene titer within the15-L bioreactor fed with glucose. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. FIG. 125C shows thetime course of total isoprene produced from the 15-L bioreactor fed withglucose. FIG. 125D shows the total carbon dioxide evolution rate (TCER),or metabolic activity profile, within the 15-L bioreactor fed withglucose.

FIGS. 126A-126E are graphs of isoprene production by E. coli strain(EWL256) expressing genes from the MVA pathway and grown in fed-batchculture at the 15-L scale with yeast extract feeding. FIG. 126A showsthe time course of optical density within the 15-L bioreactor fed withglucose. FIG. 126B shows the time course of isoprene titer within the15-L bioreactor fed with glucose. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. FIG. 126C shows thetime course of total isoprene produced from the 15-L bioreactor fed withglucose. FIG. 126D shows the volumetric productivity within the 15-Lbioreactor fed with glucose. An average value of 1.1 g/L/hr wasmaintained for a 40-hour period (23-63 hours) with yeast extractfeeding. FIG. 126E shows the carbon dioxide evolution rate (CER), ormetabolic activity profile, within the 15-L bioreactor fed with glucose.

FIGS. 127A-127D shows production of isoprene from different carbonsources via the MVA (pathway). FIG. 127A shows growth of E. coli EWL256,which contains both the MVA pathway and isoprene synthase, on eitherglucose, biomass hydrolysate, glycerol, or acetate as the only carbonsource. The different carbon sources were added to a concentration of 1%in the media. A negative control with no added carbon source wasincluded. Growth was measured as optical density at 600 nM. FIG. 127Bshows specific productivity of isoprene from E. coli EWL256 containingboth the MVA pathway and isoprene synthase when grown on either glucose,biomass hydrolysate, glycerol, or acetate as only carbon source. Thedifferent carbon sources were added to a concentration of 1% in themedia. A negative control with no added carbon source was included.Samples were taken 190 minutes, 255 minutes and 317 minutes afterinoculation and isoprene produced by the bacteria was measured usingGC-MS. FIG. 127C shows growth of E. coli EWL256 on either glucose orxylose as the only carbon source. The different carbon sources wereadded to a concentration of 1% in the media. A negative control with noadded carbon source was included. Growth was measured as optical densityat 600 nM. FIG. 127D shows specific productivity of isoprene from E.coli EWL256 when grown on either glucose or xylose as only carbonsource. The carbon sources were added to a concentration of 1% in themedia. A negative control with no added carbon source was included.Samples were taken 260 minutes, 322 minutes and 383 minutes afterinoculation and isoprene produced by the bacteria was measured usingGC-MS.

FIGS. 128A and 128B show the production of isoprene by E. coli strainsfrom glucose and from fatty acid, respectively. For FIG. 128A, elevencolonies from the transformation of WW4 with pMCM118, the plasmidbearing the lower mevalonic acid pathway, were picked to verify thepresence of the lower pathway. Cell from the colonies were cultured inTM3 medium containing 0.1% yeast extract and 2% glucose. Aliquots ofinduced culture were assayed for isoprene production after 4 hours ofinduction. All colonies showed the production of isoprene. The inducerIPTG had a strong growth inhibitory effect as was evident from the 3 to4.6-fold reduced cell density in going from 50 to 900 μM concentrationof the inducer (data not shown). The graph shows that higher induction,yields a higher specific titer of isoprene. For FIG. 128B, theproduction culture was inoculated from a washed overnight culture at 1to 10 dilution. The culture was grown for several hours and induced with50 μM IPTG. The left bar shows isoprene assay results four hours afterinduction followed by a one hour isoprene accumulation assay. The middlebar shows the one hour normalized value for the same culture with thesame induction period but analyzed by a 12 hour isoprene accumulationassay. The right bar shows the value for a one hour isopreneaccumulation assay of the culture that was induced for 13 hours.

FIG. 129 is a map of the E. coli-Streptomyces shuttle vector pUWL201PW(6400 bp) used for cloning isoprene synthase from Kudzu. Tsr,thiostrepton resistance gene. Picture is taken from Doumith et al., Mol.Gen. Genet. 264: 477-485, 2000.

FIG. 130 shows isoprene formation by Streptomyces albus wild type strain(“wt”) and strains harboring plasmid pUWL201PW (negative control) orpUWL201_iso (encoding isoprene synthase from Kudzu).

FIG. 131A is a map of the M. mazei archaeal Lower Pathway operon.

FIG. 131B depict the first portion of the nucleotide sequence of the M.mazei archaeal lower Pathway operon (SEQ ID NO:127).

FIG. 131C depict the final portion of the nucleotide sequence of the M.mazei archaeal lower Pathway operon which follows the sequence shown inFIG. 131B.

FIG. 132A is a map of MCM376-MVK from M. mazei archaeal Lowerin pET200D.

FIG. 132B depicts the first portion of the nucleotide sequence ofMCM376-MVK from M. mazei archaeal Lowerin pET200D (SEQ ID NO:128).

FIG. 132C depicts the final portion of the nucleotide sequence ofMCM376-MVK from M. mazei archaeal Lowerin pET200D which follows thesequence shown in FIG. 132B.

FIGS. 133A-133D show growth and specific productivity of isopreneproduction for EWL256 compared to RM11608-2. Growth (OD550) isrepresented by the white diamonds; specific productivity of isoprene isrepresented by the solid bars. The x-axis is time (hours) post-inductionwith either 200 (FIGS. 133A and 133B) or 400 (FIGS. 133C and 133D) μMIPTG. Y-1 axis is productivity of isoprene (μg/L/OD/hr) and Y-2 isarbitrary units of optical density at a wavelength of 550. These valuesfor the OD550 must be multiplied by 6.66 to obtain the actual OD of theculture.

FIG. 134 is a map of plasmid pBBRCMPGI1.5-pgl.

FIG. 135A depicts the first portion of the nucleotide sequence ofplasmid pBBRCMPGI1.5-pgl (SEQ ID NO:136).

FIG. 135B depicts the final portion of the nucleotide sequence ofplasmid pBBRCMPGI1.5-pgl which follows the sequence shown in FIG. 135A.

FIGS. 136A-136F are graphs of isoprene production by E. coli strainexpressing M. mazei mevalonate kinase, P. alba isoprene synthase, andpgl (RHM111608-2), and grown in fed-batch culture at the 15-L scale.FIG. 136A shows the time course of optical density within the 15-Lbioreactor fed with glucose. FIG. 136B shows the time course of isoprenetiter within the 15-L bioreactor fed with glucose. The titer is definedas the amount of isoprene produced per liter of fermentation broth.Method for calculating isoprene: cumulative isoprene produced in 59 hrs,g/Fermentor volume at 59 hrs, L [=] g/L broth. FIG. 136C also shows thetime course of isoprene titer within the 15-L bioreactor fed withglucose. Method for calculating isoprene: ∫(Instantaneous isopreneproduction rate, g/L/hr) dt from t=0 to 59 hours [=] g/L broth. FIG.136D shows the time course of total isoprene produced from the 15-Lbioreactor fed with glucose. FIG. 136E shows volumetric productivitywithin the 15-L bioreactor fed with glucose. FIG. 136F shows carbondioxide evolution rate (CER), or metabolic activity profile, within the15-L bioreactor fed with glucose.

FIG. 137A is a map of plasmid pJ201:19813.

FIG. 137B depicts the first portion of the nucleotide sequence ofpJ201:19813 (SEQ ID NO:137).

FIG. 137C depicts the final portion of the nucleotide sequence ofpJ201:19813 which follows the sequence shown in FIG. 137B.

FIG. 138 shows the time course of optical density within the 15-Lbioreactor fed with glucose.

FIG. 139 shows the time course of isoprene titer within the 15-Lbioreactor fed with glucose. The titer is defined as the amount ofisoprene produced per liter of fermentation broth.

FIG. 140 shows the time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 141 is a graph illustrating the time course of optical densitywithin the 500-L bioreactor fed with glucose and yeast extract.

FIG. 142 is a graph illustrating the time course of isoprene titerwithin the 500-L bioreactor fed with glucose and yeast extract. Thetiter is defined as the amount of isoprene produced per liter offermentation broth.

FIG. 143 is a graph illustrating the time course of total isopreneproduced form the 500-L bioreactor fed with glucose and yeast extract.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, inter alia, compositions and methods forproducing a polymer of isoprene from renewable resources. In oneembodiment, provided herein are compositions and methods for makingcopolymers of isoprene and other non-isoprene molecules. In anotherembodiment, provided herein is a polymer of isoprene derived fromrenewable resources of various molecular weights, for example, acis-1,4-polyisoprene homopolymer rubber. The polymer is produced bypolymerizing isoprene derived from renewable resources. The syntheticisoprene containing polymers of this invention offer the benefit ofbeing verifiable as to being derived from non-petrochemical basedresources. In one aspect, the isoprene from renewable resourcescomprises isoprene from bioisoprene compositions. In another aspect,isoprene derived from renewable resources can be isoprene frombioisoprene compositions. In another aspect, the isoprene derived fromrenewable resources can be a bioisoprene composition produced byculturing cells expressing a heterologous isoprene synthase enzyme. Insome aspects, the isoprene derived from renewable resources undergoespolymerization to produce polyisoprene such as cis-1,4-polyisoprene. Inother aspects, the isoprene derived from renewable resources undergoespolymerization with one or more of other monomers to produce co-polymerscomprising repeating units that are derived from isoprene monomer.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although any methodsand materials similar or equivalent to those described herein find usein the practice of the present invention, the preferred methods andmaterials are described herein. Accordingly, the terms definedimmediately below are more fully described by reference to theSpecification as a whole. All documents cited are, in relevant part,incorporated herein by reference. However, the citation of any documentis not to be construed as an admission that it is prior art with respectto the present invention.

As used herein, “renewable resources” refers to resources that are notfossil fuels. Generally, renewable resources are derived from livingorganisms or recently living organisms that can be replenished as theyare consumed. Renewable resources can be replaced by natural ecologicalcycles or sound management practices. Non-limiting examples includebiomass (e.g., switchgrass, hemp, corn, poplar, willow, sorghum,sugarcane), trees, and other plants. Renewable resources, renewablecarbon sources and bio-renewable resources are generally interchangeableherein.

As used herein, “at least a portion of the isoprene startingcomposition” can refer to at least about 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%,99.9%, or 100% of the isoprene starting composition undergoingpolymerization.

The term “isoprene” or “isoprene monomer” refers to2-methyl-1,3-butadiene (CAS#78-79-5), which is the direct and finalvolatile C5 hydrocarbon product from the elimination of pyrophosphatefrom 3,3-dimethylallyl pyrophosphate (DMAPP), and does not involve thelinking or polymerization of [an] IPP molecule(s) to [a] DMAPPmolecule(s). The term “isoprene” is not generally intended to be limitedto its method of production unless indicated otherwise herein.

As used herein, “biologically produced isoprene” or “bioisoprene” isisoprene produced by any biological means, such as produced bygenetically engineered cell cultures, natural microbials, plants oranimals.

A “bioisoprene composition” refers to a composition that can be producedby biological mean, such as systems (e.g., cells) that are engineered toproduce isoprene. It contains isoprene and other compounds that areco-produced (including impurities) and/or isolated together withisoprene. A bioisoprene composition usually contains fewer hydrocarbonimpurities than isoprene produced from petrochemical sources and oftenrequires minimal treatment in order to be of polymerization grade. Abioisoprene composition also has a different impurity profile from apetrochemically produced isoprene composition.

Bioisoprene derived from renewable carbon can be converted to a varietyof polymers by chemical polymerization. Provided herein are methods forrecovering isoprene from fermentation and subsequent conversion topolymers comprising repeating units that are derived from isoprenemonomer. These methods include, but are not limited to, recovering andpurifying isoprene from fermentation off-gas and subsequent gas orliquid phase polymerization. Both continuous and batch mode processesare contemplated within the scope of the invention.

As further detailed herein, bioisoprene compositions are distinguishedfrom petroleum-based isoprene (herein referred to as “petro-isoprene”)compositions in that bioisoprene compositions are substantially free ofany contaminating unsaturated C5 hydrocarbons that are usually presentin petro-isoprene compositions, such as, but not limited to,1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne. Ifany contaminating unsaturated C5 hydrocarbons are present in thebioisoprene starting material composition described herein, they arepresent in lower levels than that in petro-isoprene compositions.Several of these impurities are particularly problematic given theirstructural similarity to isoprene and the fact that they can act aspolymerization catalyst poisons. As detailed below, biologicallyproduced isoprene compositions can be substantially free of anycontaminating unsaturated C5 hydrocarbons without undergoing extensivepurification.

Bioisoprene composition is distinguished from petro-isoprene compositionin that bioisoprene composition contains other bio-byproducts (compoundsderived from the biological sources and/or associated the biologicalprocesses that are obtained together with bioisoprene) that are notpresent or present in much lower levels in petro-isoprene compositions,such as alcohols, aldehydes, ketone and the like. The bio-byproducts mayinclude, but are not limited to, ethanol, acetone, methanol,acetaldehyde, methacrolein, methyl vinyl ketone,2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,2,3-cycloheptenolpyridine, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene,geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol) or a linear isoprene polymer (such as alinear isoprene dimer or a linear isoprene trimer derived from thepolymerization of multiple isoprene units). In some aspects, one or moreof these compounds are removed from the bioisoprene composition prior topolymerization. In other aspects, one of more of these compounds areincluded in the polymerization reaction.

Further, bioisoprene is distinguished from petro-isoprene by carbonfinger-printing. In one aspect, bioisoprene has a higher radioactivecarbon-14 (¹⁴C) content or higher ¹⁴C/¹²C ratio that petro-isoprene.Bioisoprene is produced from renewable carbon sources, thus the ¹⁴Ccontent or the ¹⁴C/¹²C ratio in bio-isoprene is the same as that in thepresent atmosphere. Petro-isoprene, on the other hand, is derived fromfossil fuels deposited thousands to millions of years ago, thus the ¹⁴Ccontent or the ¹⁴C/¹²C ratio is diminished due to radioactive decay. Asdiscussed in greater detail herein, the fuel products derived frombioisoprene has higher ¹⁴C content or ¹⁴C/¹²C ratio than fuel productsderived from petro-isoprene. In one embodiment, a fuel product derivedfrom bioisoprene described herein has a ¹⁴C content or ¹⁴C/¹²C ratiosimilar to that in the atmosphere. In another aspect, bioisoprene can beanalytically distinguished from petro-isoprene by the stable carbonisotope ration (¹³C/¹²C), which can be reported as “delta values”represented by the symbol δ¹³C. For examples, for isoprene derived fromextractive distillation of C₅ streams from petroleum refineries, δ¹³C isabout −22‰ to about −24‰. This range is typical for light, unsaturatedhydrocarbons derived from petroleum, and products derived frompetroleum-based isoprene typically contain isoprenic units with the sameδ¹³C. Bioisoprene produced by fermentation of corn-derived glucose (δ¹³C−10.73%) with minimal amounts of other carbon-containing nutrients(e.g., yeast extract) produces isoprene which can be polymerized intopolyisoprene with δ¹³C −14.66‰ to −14.85‰. Products produced from suchbioisoprene are expected to have δ¹³C values that are less negative thanthose derived from petroleum-based isoprene.

While isoprene can be obtained by fractionating petroleum, thepurification of this material is expensive and time-consuming. Petroleumcracking of the C5 stream of hydrocarbons produces only about 15%isoprene. Isoprene is also naturally produced by a variety of microbial,plant, and animal species. In particular, two pathways have beenidentified for the biosynthesis of isoprene: the mevalonate (MVA)pathway and the non-mevalonate (DXP) pathway. Genetically engineeredcell cultures in bioreactors have produced isoprene more efficiently, inlarger quantities, in higher purities and/or with unique impurityprofiles, e.g. as described in U.S. provisional patent application Nos.61/013,386 and 61/013,574, filed on Dec. 13, 2007, WO 2009/076676, U.S.provisional patent application Nos. 61/134,094, 61/134,947, 61/134,011and 61/134,103, filed on Jul. 2, 2008, WO 2010/003007, U.S. provisionalpatent application No. 61/097,163, filed on Sep. 15, 2008, WO2010/031079, U.S. provisional patent application No. 61/097,186, filedon Sep. 15, 2008, WO 2010/031062, U.S. provisional patent applicationNo. 61/097,189, filed on Sep. 15, 2008, WO 2010/031077, U.S. provisionalpatent application No. 61/097,200, filed on Sep. 15, 2008, WO2010/031068, U.S. provisional patent application No. 61/097,204, filedon Sep. 15, 2008, WO 2010/031076, U.S. provisional patent applicationNo. 61/141,652, filed on Dec. 30, 2008, PCT/US09/069,862, U.S. patentapplication Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102A1) and U.S. patent application Ser. No. 12/429,143, filed Apr. 23, 2009(US 2010/0003716 A1), which are incorporated by reference in theirentireties.

In one aspect, the invention features compositions and systems forproducing a polymer of isoprene comprising: (a) an isoprene startingcomposition derived from renewable resources; and (b) a polymer producedfrom at least a portion of the isoprene starting material; where atleast a portion of the isoprene starting composition undergoespolymerization. An isoprene starting material derived from renewableresources is subjected to chemical polymerization to produce a polymercomprising repeating units that are derived from isoprene monomer fromrenewable sources. In one aspect, an isoprene starting compositionderived from renewable resources can be a bioisoprene compositionderived from renewable carbon sources.

Exemplary Starting Isoprene Compositions

In some embodiments, the isoprene starting composition derived fromrenewable resources comprises greater than or about 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1000 mg of isoprene. In some embodiments, the starting isoprenecomposition comprises greater than or about 2, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100 g of isoprene. In some embodiments, the startingisoprene composition comprises greater than or about 0.2, 0.5, 1, 2, 5,10, 20, 50, 100, 200, 500, 1000 kg of isoprene. In some embodiments, theamount of isoprene in the starting composition is between about 2 toabout 5,000 mg, such as between about 2 to about 100 mg, about 100 toabout 500 mg, about 500 to about 1,000 mg, about 1,000 to about 2,000mg, or about 2,000 to about 5,000 mg. In some embodiments, the amount ofisoprene in the starting composition is between about 20 to about 5,000mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg, about 200to about 1,000 mg, about 300 to about 1,000 mg, or about 400 to about1,000 mg. In some embodiments, the amount of isoprene in the startingcomposition is between about 2 to about 5,000 g, such as between about 2to about 100 g, about 100 to about 500 g, about 500 to about 1,000 g,about 1,000 to about 2,000 g, or about 2,000 to about 5,000 g. In someembodiments, the amount of isoprene in the starting composition isbetween about 2 to about 5,000 kg, about 10 to about 2,000 kg, about 20to about 1,000 kg, about 20 to about 500 kg, about 30 to about 200 kg,or about 40 to about 100 kg. In some embodiments, greater than or about20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% (w/w) of the volatile organicfraction of the starting composition is isoprene.

In some embodiments, the isoprene starting composition derived fromrenewable resources comprises greater than or about 98.0, 98.5, 99.0,99.5, or 100% isoprene by weight compared to the total weight of all C5hydrocarbons in the starting composition. In some embodiments, thehighly pure isoprene starting composition comprises greater than orabout 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the startingcomposition. In some embodiments, the starting composition has arelative detector response of greater than or about 98.0, 98.5, 99.0,99.5, or 100% for isoprene compared to the detector response for all C5hydrocarbons in the starting composition. In some embodiments, thestarting composition has a relative detector response of greater than orabout 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98,99.99, or 100% for isoprene compared to the detector response for all C5hydrocarbons in the starting composition. In some embodiments, thestarting isoprene composition comprises between about 98.0 to about98.5, about 98.5 to about 99.0, about 99.0 to about 99.5, about 99.5 toabout 99.8, about 99.8 to 100% isoprene by weight compared to the totalweight of all C5 hydrocarbons in the starting composition. In someembodiments, the starting isoprene composition comprises between about99.90 to about 99.92, about 99.92 to about 99.94, about 99.94 to about99.96, about 99.96 to about 99.98, about 99.98 to 100% isoprene byweight compared to the total weight of all C5 hydrocarbons in thestarting composition.

In some embodiments, the isoprene starting composition derived fromrenewable resources comprises less than or about 2.0, 1.5, 1.0, 0.5,0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005,0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the starting composition. In some embodiments, thestarting composition has a relative detector response of less than orabout 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01,0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for C5 hydrocarbonsother than isoprene compared to the detector response for all C5hydrocarbons in the starting composition. In some embodiments, thestarting composition has a relative detector response of less than orabout 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01,0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne compared to the detector response for all C5hydrocarbons in the starting composition. In some embodiments, thehighly pure isoprene starting composition comprises between about 0.02to about 0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about0.08 to 0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other thanisoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene,trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene,2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared tothe total weight of all C5 hydrocarbons in the starting composition.

In some embodiments, the isoprene starting composition derived fromrenewable resources comprises less than or about 50, 40, 30, 20, 10, 5,1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits thepolymerization of isoprene for any compound in the starting compositionthat inhibits the polymerization of isoprene. In some embodiments, thestarting isoprene composition comprises between about 0.005 to about 50,such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 toabout 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 μg/L of acompound that inhibits the polymerization of isoprene for any compoundin the starting composition that inhibits the polymerization ofisoprene. In some embodiments, the starting isoprene compositioncomprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05,0.01, or 0.005 μg/L of a hydrocarbon other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne). In some embodiments, the starting isoprenecomposition comprises between about 0.005 to about 50, such as about0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about0.01 to about 0.5, or about 0.01 to about 0.005 μg/L of a hydrocarbonother than isoprene. In some embodiments, the starting isoprenecomposition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5,0.1, 0.05, 0.01, or 0.005 μg/L of a protein or fatty acid (such as aprotein or fatty acid that is naturally associated with natural rubber).

In some embodiments, the isoprene starting composition derived fromrenewable resources comprises less than or about 10, 5, 1, 0.8, 0.5,0.1, 0.05, 0.01, or 0.005 ppm of alpha acetylenes, piperylenes,acetonitrile, or 1,3-cyclopentadiene. In some embodiments, the startingisoprene composition comprises less than or about 5, 1, 0.5, 0.1, 0.05,0.01, or 0.005 ppm of sulfur or allenes. In some embodiments, thestarting isoprene composition comprises less than or about 30, 20, 15,10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes (such as1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne). In some embodiments,the starting isoprene composition comprises less than or about 2000,1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or0.005 ppm of isoprene dimers, such as cyclic isoprene dimmers (e.g.,cyclic C10 compounds derived from the dimerization of two isopreneunits).

In some embodiments, the isoprene starting composition derived fromrenewable resources includes ethanol, acetone, methanol, acetaldehyde,methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more ofthe foregoing. In particular embodiments, the starting isoprenecomposition comprises greater than or about 0.005, 0.01, 0.05, 0.1, 0.5,1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone,methanol, acetaldehyde, methacrolein, methyl vinyl ketone,2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),or any two or more of the foregoing. In some embodiments, the isoprenecomposition comprises between about 0.005 to about 120, such as about0.01 to about 80, about 0.01 to about 60, about 0.01 to about 40, about0.01 to about 30, about 0.01 to about 20, about 0.01 to about 10, about0.1 to about 80, about 0.1 to about 60, about 0.1 to about 40, about 5to about 80, about 5 to about 60, or about 5 to about 40 μg/L ofethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinylketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene,a C5 prenyl alcohol, or any two or more of the foregoing.

In some embodiments, the isoprene starting composition derived fromrenewable resources includes one or more of the following components:2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as alinear isoprene dimer or a linear isoprene trimer derived from thepolymerization of multiple isoprene units). In various embodiments, theamount of one of these components relative to amount of isoprene inunits of percentage by weight (i.e., weight of the component divided bythe weight of isoprene times 100) is greater than or about 0.01, 0.02,0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%(w/w). In some embodiments, the relative detector response for thesecond compound compared to the detector response for isoprene isgreater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, or 110%. In various embodiments, the amount ofone of these components relative to amount of isoprene in units ofpercentage by weight (i.e., weight of the component divided by theweight of isoprene times 100) is between about 0.01 to about 105% (w/w),such as about 0.01 to about 90, about 0.01 to about 80, about 0.01 toabout 50, about 0.01 to about 20, about 0.01 to about 10, about 0.02 toabout 50, about 0.05 to about 50, about 0.1 to about 50, or 0.1 to about20% (w/w).

In some embodiments, at least a portion of the isoprene startingcomposition derived from renewable resources is in a gas phase. In someembodiments, at least a portion of the isoprene starting compositionderived from renewable resources is in a liquid phase (such as acondensate). In some embodiments, at least a portion of the isoprenestarting composition derived from renewable resources is in a solidphase. In some embodiments, at least a portion of the isoprene startingcomposition derived from renewable resources is absorbed to a solidsupport, such as a support that includes silica and/or activated carbon.In some embodiments, the starting isoprene composition is mixed with oneor more solvents. In some embodiments, the starting isoprene compositionis mixed with one or more gases.

Techniques for producing isoprene in cultures of cells that produceisoprene are described in U.S. provisional patent application Nos.61/013,386 and 61/013,574, filed on Dec. 13, 2007, WO 2009/076676, U.S.provisional patent application Nos. 61/134,094, 61/134,947, 61/134,011and 61/134,103, filed on Jul. 2, 2008, WO 2010/003007, U.S. provisionalpatent application No. 61/097,163, filed on Sep. 15, 2008, WO2010/031079, U.S. provisional patent application No. 61/097,186, filedon Sep. 15, 2008, WO 2010/031062, U.S. provisional patent applicationNo. 61/097,189, filed on Sep. 15, 2008, WO 2010/031077, U.S. provisionalpatent application No. 61/097,200, filed on Sep. 15, 2008, WO2010/031068, U.S. provisional patent application No. 61/097,204, filedon Sep. 15, 2008, WO 2010/031076, U.S. provisional patent applicationNo. 61/141,652, filed on Dec. 30, 2008, PCT/US09/069,862, U.S. patentapplication Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102A1) and U.S. patent application Ser. No. 12/429,143, filed Apr. 23, 2009(US 2010/0003716 A1), the teachings of which are incorporated herein byreference for the purpose of teaching techniques for producing andrecovering isoprene by such a process. In any case, U.S. provisionalpatent application Nos. 61/013,386 and 61/013,574, filed on Dec. 13,2007, WO 2009/076676, U.S. provisional patent application Nos.61/134,094, 61/134,947, 61/134,011 and 61/134,103, filed on Jul. 2,2008, WO 2010/003007, U.S. provisional patent application No.61/097,163, filed on Sep. 15, 2008, WO 2010/031079, U.S. provisionalpatent application No. 61/097,186, filed on Sep. 15, 2008, WO2010/031062, U.S. provisional patent application No. 61/097,189, filedon Sep. 15, 2008, WO 2010/031077, U.S. provisional patent applicationNo. 61/097,200, filed on Sep. 15, 2008, WO 2010/031068, U.S. provisionalpatent application No. 61/097,204, filed on Sep. 15, 2008, WO2010/031076, U.S. provisional patent application No. 61/141,652, filedon Dec. 30, 2008, PCT/US09/069,862, U.S. patent application Ser. No.12/335,071, filed Dec. 15, 2008 (US 2009/0203102 A1) and U.S. patentapplication Ser. No. 12/429,143, filed Apr. 23, 2009 (US 2010/0003716A1) teach compositions and methods for the production of increasedamounts of isoprene in cell cultures. U.S. patent application Ser. No.12/335,071, filed Dec. 15, 2008 (US 2009/0203102 A1) further teachescompositions and methods for co-production of isoprene and hydrogen fromcultured cells. In particular, these compositions and methodscompositions and methods increase the rate of isoprene production andincrease the total amount of isoprene that is produced. For example,cell culture systems that generate 4.8×10⁴ nmole/g_(wcm)/hr of isoprenehave been produced (Table 1). The efficiency of these systems isdemonstrated by the conversion of about 2.2% of the carbon that thecells consume from a cell culture medium into isoprene. As shown in theExamples and Table 2, approximately 3 g of isoprene per liter of brothwas generated. If desired, even greater amounts of isoprene can beobtained using other conditions, such as those described herein. In someembodiments, a renewable carbon source is used for the production ofisoprene. In some embodiments, the production of isoprene is decoupledfrom the growth of the cells. In some embodiments, the concentrations ofisoprene and any oxidants are within the nonflammable ranges to reduceor eliminate the risk that a fire may occur during production orrecovery of isoprene. The compositions and methods are desirable becausethey allow high isoprene yield per cell, high carbon yield, highisoprene purity, high productivity, low energy usage, low productioncost and investment, and minimal side reactions. This efficient, largescale, biosynthetic process for isoprene production provides an isoprenesource for synthetic isoprene-based products such as rubber and providesa desirable, low-cost alternative to using natural rubber.

As discussed further below, the amount of isoprene produced by cells canbe greatly increased by introducing a heterologous nucleic acid encodingan isoprene synthase polypeptide (e.g., a plant isoprene synthasepolypeptide) into the cells. Isoprene synthase polypeptides convertdimethylallyl diphosphate (DMAPP) into isoprene. As shown in theExamples, a heterologous Pueraria Montana (kudzu) or Populus alba(Poplar) isoprene synthase polypeptide was expressed in a variety ofhost cells, such as Escherichia coli, Panteoa citrea, Bacillus subtilis,Yarrowia lipolytica, and Trichoderma reesei. As also shown in theExamples, a heterologous Methanosarcina mazei (M. mazei) mevalonatekinase (MVK) was expressed in host cells such as Escherichia coli toincrease isoprene production. All of these cells produced more isoprenethan the corresponding cells without the heterologous isoprene synthasepolypeptide. As illustrated in Tables 1 and 2, large amounts of isopreneare produced using the methods described herein. For example, B.subtilis cells with a heterologous isoprene synthase nucleic acidproduced approximately 10-fold more isoprene in a 14 liter fermentorthan the corresponding control B. subtilis cells without theheterologous nucleic acid (Table 2). The production of 60.5 g ofisoprene per liter of broth (mg/L, wherein the volume of broth includesboth the volume of the cell medium and the volume of the cells) by E.coli and 30 mg/L by B. subtilis in fermentors indicates that significantamounts of isoprene can be generated (Table 2). If desired, isoprene canbe produced on an even larger scale or other conditions described hereincan be used to further increase the amount of isoprene. The vectorslisted in Tables 1 and 2 and the experimental conditions are describedin further detail below and in the Examples section.

TABLE 1 Exemplary yields of isoprene from a shake flask using the cellcultures and methods of the invention. The assay for measuring isopreneproduction is described in Example I, part II. For this assay, a samplewas removed at one or more time points from the shake flask and culturedfor 30 minutes. The amount of isoprene produced in this sample was thenmeasured. The headspace concentration and specific rate of isopreneproduction are listed in Table 1 and described further herein. IsopreneProduction in a Headspace vial* Headspace Specific Rate concentrationμg/L_(broth)/hr/OD Strain μg/L_(gas) (nmol/g_(wcm)/hr) E. coliBL21/pTrcKudzu IS 1.40  53.2 (781.2) E. coli BL21/Pcl DXS yidi 7.61289.1 Kudzu IS (4.25 × 10³) E. coli BL21/MCM127 with 23.0 874.1 kudzu ISand entire MVA (1.28 × 10⁴) pathway E. coli BL21/Pet N- 1.49  56.6HisKudzu IS (831.1) Pantoea citrea/pTrcKudzu 0.66  25.1 IS (368.6) E.coli w/ Poplar IS —  5.6 [Miller (2001)]  (82.2) Bacillis licheniformisFall —  4.2 U.S. Pat. No. 5,849,970  (61.4) Yarrowia lipolytica with~0.05 μg/L ~2  kudzu isoprene synthase (~30)   Trichoderma reesei with~0.05 μg/L ~2  kudzu isoprene synthase (~30)   E. coli BL21/ 85.9  3.2 ×10³ pTrcKKD_(y)I_(k)IS with kudzu  (4.8 × 10⁴) IS and lower MVA pathway*Normalized to 1 mL of 1 OD₆₀₀, cultured for 1 hour in a sealedheadspace vial with a liquid to headspace volume ratio of 1:19.

TABLE 2 Exemplary yields of isoprene in a fermentor using the cellcultures and methods of the invention. The assay for measuring isopreneproduction is described in Example I, part II. For this assay, a sampleof the off-gas of the fermentor was taken and analyzed for the amount ofisoprene. The peak headspace concentration (which is the highestheadspace concentration during the fermentation), titer (which is thecumulative, total amount of isoprene produced per liter of broth), andpeak specific rate of isoprene production (which is the highest specificrate during the fermentation) are listed in Table 2 and describedfurther herein. Isoprene Production in Fermentors Peak Headspace PeakSpecific rate concentration** Titer μg/L_(broth)/hr/OD Strain(μg/L_(gas)) (mg/L_(broth)) (nmol/g_(wcm)/hr) E. coli BL21/ 52 41.2 37 pTrcKudzu with (543.3) Kudzu IS E. coli 3 3.5  21.4 FM5/pTrcKudzu IS(308.1) E. coli BL21/triple 285 300 240   strain (DXS, yidi, (3.52 ×10³) IS) E. coli FM5/triple 50.8 29 180.8 strain (DXS, yidi, (2.65 ×10³) IS) E. coli/MCM127 1094 250 875   with Kudzu IS and (1.28 × 10⁴)entire MVA pathway E. coli 2418 1640 1248   BL21/pCLPtrc (1.83 × 10⁴)UpperPathway gi1.2 integrated lower pathway pTrcKudzu E. coli 3500 33001088   BL21/pCLPtrc (1.60 × 10⁴) UpperPathwayHGS2- pTrcKKDyIkIS Bacillussubtilis 1.5 2.5  0.8 wild-type  (11.7) Bacillus pBS Kudzu 16.6 ~30  5IS (over 100  (73.4) hours) Bacillus Marburg 2.04 0.61  24.5 6051[Wagner and (359.8) Fall (1999)] Bacillus Marburg 0.7 0.15  6.8 6051Fall (100)   U.S. Pat. No. 5,849,970 E. coli 2.03 × 10⁴ 3.22 × 10⁴  5.9× 10³ BL21/pCLPtrcUpper (8.66 × 10⁴) Pathway and gil.2KKDyI andpTrcAlba-mMVK E. coli 3.22 × 10⁴ 6.05 × 10⁴ 1.28 × 10⁴ BL21/pCLPtrcUpper(1.88 × 10⁵) Pathway and gi1.2KKDyI and pTrcAlba-mMVK pluspBBRCMPGI1.5pgl **Normalized to an off-gas flow rate of 1 vvm (1 volumeoff-gas per 1 L_(broth) per minute).

Additionally, isoprene production by cells that contain a heterologousisoprene synthase nucleic acid can be enhanced by increasing the amountof a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or anisopentenyl diphosphate isomerase (IDI) polypeptide expressed by thecells. For example, a DXS nucleic acid and/or an IDI nucleic acid can beintroduced into the cells. The DXS nucleic acid may be a heterologousnucleic acid or a duplicate copy of an endogenous nucleic acid.Similarly, the IDI nucleic acid may be a heterologous nucleic acid or aduplicate copy of an endogenous nucleic acid. In some embodiments, theamount of DXS and/or IDI polypeptide is increased by replacing theendogenous DXS and/or IDI promoters or regulatory regions with otherpromoters and/or regulatory regions that result in greater transcriptionof the DXS and/or IDI nucleic acids. In some embodiments, the cellscontain both a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

The encoded DXS and IDI polypeptides are part of the DXP pathway for thebiosynthesis of isoprene (FIG. 19A). DXS polypeptides convert pyruvateand D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate.While not intending to be bound by any particular theory, it is believedthat increasing the amount of DXS polypeptide increases the flow ofcarbon through the DXP pathway, leading to greater isoprene production.IDI polypeptides catalyze the interconversion of isopentenyl diphosphate(IPP) and dimethylallyl diphosphate (DMAPP). While not intending to bebound by any particular theory, it is believed that increasing theamount of IDI polypeptide in cells increases the amount (and conversionrate) of IPP that is converted into DMAPP, which in turn is convertedinto isoprene.

For example, fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used toproduce isoprene. The levels of isoprene varied from 50 to 300 μg/L overa time period of 15 hours (Example 7, part VII). As another example,fermentation of E. coli with M. mazei mevalonate kinase (MVK), P. albaisoprene synthase, the upper MVA pathway, and the integrated lower MVApathway was used to produce isoprene. The levels of isoprene varied from32 to 35.6 g/L over a time period of 67 hours (Example 14, part III).

In yet another example, fermentation of E. coli with M. mazei mevalonatekinase (MVK), P. alba isoprene synthase, pgl over-expression(RHM111608-2), the upper MVA pathway, and the integrated lower MVApathway were used to produce isoprene. The levels of isoprene vary from33.2 g/L to 40.0 g/L over a time period of 40 hours or 48.6 g/L to 60.5g/L over a time period of 59 hours (Example 17, part (ii)).

In some embodiments, the presence of heterologous or extra endogenousisoprene synthase, IDI, and DXS nucleic acids causes cells to grow morereproducibly or remain viable for longer compared to the correspondingcell with only one or two of these heterologous or extra endogenousnucleic acids. For example, cells containing heterologous isoprenesynthase, IDI, and DXS nucleic acids grew better than cells with onlyheterologous isoprene synthase and DXS nucleic acids or with only aheterologous isoprene synthase nucleic acid. Also, heterologous isoprenesynthase, IDI, and DXS nucleic acids were successfully operably linkedto a strong promoter on a high copy plasmid that was maintained by E.coli cells, suggesting that large amounts of these polypeptides could beexpressed in the cells without causing an excessive amount of toxicityto the cells. While not intending to be bound to a particular theory, itis believed that the presence of heterologous or extra endogenousisoprene synthase and IDI nucleic acids may reduce the amount of one ormore potentially toxic intermediates that would otherwise accumulate ifonly a heterologous or extra endogenous DXS nucleic acid was present inthe cells.

In some embodiments, the production of isoprene by cells that contain aheterologous isoprene synthase nucleic acid is augmented by increasingthe amount of a MVA polypeptide expressed by the cells (FIGS. 19A and19B). Exemplary MVA pathways polypeptides include any of the followingpolypeptides: acetyl-CoA acetyltransferase (AA-CoA thiolase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAreductase) polypeptides, mevalonate kinase (MVK) polypeptides,phosphomevalonate kinase (PMK) polypeptides, diphosphomevalontedecarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC)polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDIpolypeptides, and polypeptides (e.g., fusion polypeptides) having anactivity of two or more MVA pathway polypeptides. For example, one ormore MVA pathway nucleic acids can be introduced into the cells. In someembodiments, the cells contain the upper MVA pathway, which includesAA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase nucleic acids.In some embodiments, the cells contain the lower MVA pathway, whichincludes MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, thecells contain the entire MVA pathway, which includes AA-CoA thiolase,HMG-CoA synthase, HMG-CoA reductase, MVK, PMK, MVD, and IDI nucleicacids. In some embodiments, the cells contain an entire MVA pathway thatincludes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK,PMDC, IPK, and IDI nucleic acids. The MVA pathway nucleic acids may beheterologous nucleic acids or duplicate copies of endogenous nucleicacids. In some embodiments, the amount of one or more MVA pathwaypolypeptides is increased by replacing the endogenous promoters orregulatory regions for the MVA pathway nucleic acids with otherpromoters and/or regulatory regions that result in greater transcriptionof the MVA pathway nucleic acids. In some embodiments, the cells containboth a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

For example, E. coli cells containing a nucleic acid encoding a kudzuisoprene synthase polypeptide and nucleic acids encoding Saccharomycescerevisia MVK, PMK, MVD, and IDI polypeptides generated isoprene at arate of 6.67×10⁴ nmol/L_(broth)/OD₆₀₀/hr (see Example 8). Additionally,a 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid (anintermediate of the MVA pathway). A shake flask of these cells produced2-4 grams of mevalonic acid per liter. These results indicate thatheterologous MVA pathways nucleic acids are active in E. coli. E. colicells that contain nucleic acids for both the upper MVA pathway and thelower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127)produced significantly more isoprene (874

g/L_(broth)/hr/OD) compared to E. coli cells with nucleic acids for onlythe lower MVA pathway and the kudzu isoprene synthase (strain MCM 131)(see Table 3 and Example 8, part VIII). E. coli cells containing anucleic acid encoding P. alba isoprene synthase polypeptide and anucleic acid encoding M. mazei MVK polypeptide generated 320.6 g (at apeak specific rate of 9.54×10⁴ nmol/L_(broth)/OD₆₀₀/hr (i.e. 9.5×10⁻⁵mol/L_(broth)/OD₆₀₀/hr)) of isoprene during a 67 hour fermentation inthe absence of yeast extract feeding or 395.5 g (at a peak specific rateof 8.66×10⁴ nmol/L_(broth)/OD₆₀₀/hr) during a 68 hour fermentation inthe presence of yeast extract feeding (see Example 14).

In some embodiments, at least a portion of the cells maintain theheterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects of the invention,the nucleic acid comprising the heterologous or duplicate copy of anendogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acidalso comprises a selective marker, such as a kanamycin, ampicillin,carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin,or chloramphenicol antibiotic resistance nucleic acid.

As indicated in Example 7, part VI, the amount of isoprene produced canbe further increased by adding yeast extract to the cell culture mediumusing E. coli cells with kudzu isoprene synthase, S. cerevisia IDI, andE. coli DXS nucleic acids to produce isoprene. In particular, the amountof isoprene produced was linearly proportional to the amount of yeastextract in the cell medium for the concentrations tested (FIG. 48C).Additionally, approximately 0.11 grams of isoprene per liter of brothwas produced from a cell medium with yeast extract and glucose (Example7, part VIII). Increasing the amount of yeast extract in the presence ofglucose resulted in more isoprene being produced than increasing theamount of glucose in the presence of yeast extract. Also, increasing theamount of yeast extract allowed the cells to produce a high level ofisoprene for a longer length of time and improved the health of thecells.

Isoprene production was also demonstrated using three types ofhydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as thecarbon source (FIGS. 46A-C and FIGS. 127A and 127B). E. coli cells withkudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acidsproduced as much isoprene from these hydrolyzed biomass carbon sourcesas from the equivalent amount of glucose (e.g., 1% glucose, w/v). E.coli cells expressing P. alba isoprene synthase and the MVA pathwayproduced isoprene at a higher initial growth rate from ammonia fiberexpansion (AFEX) pretreated corn stover than from the equivalent amountof glucose. (FIGS. 127A and 127B). If desired, any other biomass carbonsource can be used in the compositions and methods of the invention.Biomass carbon sources are desirable because they are cheaper than manyconventional cell mediums, thereby facilitating the economicalproduction of isoprene.

Additionally, invert sugar was shown to function as a carbon source forthe generation of isoprene (FIG. 47D). For example, 2.4 g/L of isoprenewas produced from cells expressing MVA pathway polypeptides and a Kudzuisoprene synthase (Example 8, part XV). Glycerol was as also used as acarbon source for the generation of 2.2 mg/L of isoprene from cellsexpressing a Kudzu isoprene synthase (Example 8, part XIV). Expressing aDXS nucleic acid, an IDI nucleic acid, and/or one or more MVA pathwaynucleic acids (such as nucleic acids encoding the entire MVA pathway) inaddition to an isoprene synthase nucleic acid may increase theproduction of isoprene from glycerol.

Additionally, xylose, acetate, and glycerol were also shown to functionas a carbon source for the generation of isoprene (FIGS. 127A-127D). Forexample, E. coli cells with P. alba isoprene synthase and the MVApathway grown on acetate as the only carbon source had a specificproductivity of isoprene about twice as high as during growth on glucose(Example 14, Part IV; FIGS. 127A and 127B).

In some embodiments, an oil is included in the cell medium. For example,B. subtilis cells containing a kudzu isoprene synthase nucleic acidproduced isoprene when cultured in a cell medium containing an oil and asource of glucose (Example 4, part III). As another example, E. colifadR atoC mutant cells containing the upper and lower MVA pathway pluskudzu isoprene synthase produced isoprene when cultured in a cell mediumcontaining palm oil and a source of glucose (Example 16, part II). Insome embodiments, more than one oil (such as 2, 3, 4, 5, or more oils)is included in the cell medium. While not intending to be bound to anyparticular theory, it is believed that (i) the oil may increase theamount of carbon in the cells that is available for conversion toisoprene, (ii) the oil may increase the amount of acetyl-CoA in thecells, thereby increasing the carbon flow through the MVA pathway,and/or (ii) the oil may provide extra nutrients to the cells, which isdesirable since a lot of the carbon in the cells is converted toisoprene rather than other products. In some embodiments, cells that arecultured in a cell medium containing oil naturally use the MVA pathwayto produce isoprene or are genetically modified to contain nucleic acidsfor the entire MVA pathway. In some embodiments, the oil is partially orcompletely hydrolyzed before being added to the cell culture medium tofacilitate the use of the oil by the host cells.

One of the major hurdles to commercial production of small moleculessuch as isoprene in cells (e.g., bacteria) is the decoupling ofproduction of the molecule from growth of the cells. In some embodimentsfor the commercially viable production of isoprene, a significant amountof the carbon from the feedstock is converted to isoprene, rather thanto the growth and maintenance of the cells (“carbon efficiency”). Invarious embodiments, the cells convert greater than or about 0.0015,0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5,4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture mediuminto isoprene. In particular embodiments, a significant portion of thecarbon from the feedstock that is converted to downstream products isconverted to isoprene. As described further in Example 11, E. coli cellsexpressing MVA pathway and kudzu isoprene synthase nucleic acidsexhibited decoupling of the production of isoprene or the intermediatemevalonic acid from growth, resulting in high carbon efficiency. Inparticular, mevalonic acid was formed from cells expressing the upperMVA pathway from Enterococcus faecalis. Isoprene was formed from cellsexpressing the upper MVA pathway from Enterococcus faecalis, the lowerMVA pathway from Saccharomyces cerevisiae, and the isoprene synthasefrom Pueraria montana (Kudzu). This decoupling of isoprene or mevalonicacid production from growth was demonstrated in four different strainsof E. coli: BL21(LDE3), BL21(LDE3) Tuner, FM5, and MG1655. The first twoE. coli strains are B strains, and the latter two are K12 strains.Decoupling of production from growth was also demonstrated in a variantof MG1655 with ack and pta genes deleted. This variant also demonstratedless production of acetate.

Exemplary Polypeptides and Nucleic Acids

Various isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides andnucleic acids can be used in the compositions and methods of theinvention.

As used herein, “polypeptides” includes polypeptides, proteins,peptides, fragments of polypeptides, and fusion polypeptides thatinclude part or all of a first polypeptide (e.g., an isoprene synthase,DXS, IDI, or MVA pathway polypeptide) and part or all of a secondpolypeptide (e.g., a peptide that facilitates purification or detectionof the fusion polypeptide, such as a His-tag). In some embodiments, thefusion polypeptide has an activity of two or more MVA pathwaypolypeptides (such as AA-CoA thiolase and HMG-CoA reductasepolypeptides). In some embodiments, the polypeptide is anaturally-occurring polypeptide (such as the polypeptide encoded by anEnterococcus faecalis mvaE nucleic acid) that has an activity of two ormore MVA pathway polypeptides.

In various embodiments, a polypeptide has at least or about 50, 100,150, 175, 200, 250, 300, 350, 400, or more amino acids. In someembodiments, the polypeptide fragment contains at least or about 25, 50,75, 100, 150, 200, 300, or more contiguous amino acids from afull-length polypeptide and has at least or about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of acorresponding full-length polypeptide. In particular embodiments, thepolypeptide includes a segment of or the entire amino acid sequence ofany naturally-occurring isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide. In some embodiments, the polypeptide has one or moremutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

In some embodiments, the polypeptide is an isolated polypeptide. As usedherein, an “isolated polypeptide” is not part of a library ofpolypeptides, such as a library of 2, 5, 10, 20, 50 or more differentpolypeptides and is separated from at least one component with which itoccurs in nature. An isolated polypeptide can be obtained, for example,by expression of a recombinant nucleic acid encoding the polypeptide.

In some embodiments, the polypeptide is a heterologous polypeptide. By“heterologous polypeptide” is meant a polypeptide whose amino acidsequence is not identical to that of another polypeptide naturallyexpressed in the same host cell. In particular, a heterologouspolypeptide is not identical to a wild-type polypeptide that is found inthe same host cell in nature.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides in either single ordouble-stranded form. In some embodiments, the nucleic acid is arecombinant nucleic acid. By “recombinant nucleic acid” means a nucleicacid of interest that is free of one or more nucleic acids (e.g., genes)which, in the genome occurring in nature of the organism from which thenucleic acid of interest is derived, flank the nucleic acid of interest.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, ora cDNA fragment produced by PCR or restriction endonuclease digestion)independent of other sequences. In various embodiments, a nucleic acidis a recombinant nucleic acid. In some embodiments, an isoprenesynthase, efe, DXS, IDI, or MVA pathway nucleic acid is operably linkedto another nucleic acid encoding all or a portion of another polypeptidesuch that the recombinant nucleic acid encodes a fusion polypeptide thatincludes an isoprene synthase, efe, DXS, IDI, or MVA pathway polypeptideand all or part of another polypeptide (e.g., a peptide that facilitatespurification or detection of the fusion polypeptide, such as a His-tag).In some embodiments, part or all of a recombinant nucleic acid ischemically synthesized. It is to be understood that mutations, includingsingle nucleotide mutations, can occur within a nucleic acid as definedherein.

In some embodiments, the nucleic acid is a heterologous nucleic acid. By“heterologous nucleic acid” is meant a nucleic acid whose nucleic acidsequence is not identical to that of another nucleic acid naturallyfound in the same host cell.

In particular embodiments, the nucleic acid includes a segment of or theentire nucleic acid sequence of any naturally-occurring isoprenesynthase, DXS, IDI, or MVA pathway nucleic acid. In some embodiments,the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400,500, 600, 700, 800, or more contiguous nucleotides from anaturally-occurring isoprene synthase nucleic acid DXS, IDI, or MVApathway nucleic acid. In some embodiments, the nucleic acid has one ormore mutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathway nucleicacid. In some embodiments, the nucleic acid has one or more mutations(e.g., a silent mutation) that increase the transcription or translationof isoprene synthase, DXS, IDI, or MVA pathway nucleic acid. In someembodiments, the nucleic acid is a degenerate variant of any nucleicacid encoding an isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a nucleic acid for improved expression in a host cell, itis desirable in some embodiments to design the nucleic acid such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell.

The accession numbers of exemplary isoprene synthase, DXS, IDI, and/orMVA pathway polypeptides and nucleic acids are listed in Appendix 1 (theaccession numbers of Appendix 1 and their corresponding sequences areherein incorporated by reference in their entireties, particularly withrespect to the amino acid and nucleic acid sequences of isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids).The Kegg database also contains the amino acid and nucleic acidsequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVApathway polypeptides and nucleic acids (see, for example, the world-wideweb at “genome.jp/kegg/pathway/map/map00100.html” and the sequencestherein, which are each hereby incorporated by reference in theirentireties, particularly with respect to the amino acid and nucleic acidsequences of isoprene synthase, DXS, IDI, and/or MVA pathwaypolypeptides and nucleic acids). In some embodiments, one or more of theisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/ornucleic acids have a sequence identical to a sequence publicly availableon Dec. 12, 2007 or Dec. 11, 2008, such as any of the sequences thatcorrespond to any of the accession numbers in Appendix 1 or any of thesequences present in the Kegg database. Additional exemplary isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acidsare described further below.

Exemplary Isoprene Synthase Polypeptides and Nucleic Acids

As noted above, isoprene synthase polypeptides convert dimethylallyldiphosphate (DMAPP) into isoprene. Exemplary isoprene synthasepolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an isoprenesynthase polypeptide. Standard methods can be used to determine whethera polypeptide has isoprene synthase polypeptide activity by measuringthe ability of the polypeptide to convert DMAPP into isoprene in vitro,in a cell extract, or in vivo. In an exemplary assay, cell extracts areprepared by growing a strain (e.g., the E. coli/pTrcKudzu straindescribed herein) in the shake flask method as described in Example 1.After induction is complete, approximately 10 mLs of cells are pelletedby centrifugation at 7000×g for 10 minutes and resuspended in 5 ml ofPEB without glycerol. The cells are lysed using a French Pressure cellusing standard procedures. Alternatively the cells are treated withlysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thawat −80 C.

Isoprene synthase polypeptide activity in the cell extract can bemeasured, for example, as described in Silver et al., J. Biol. Chem.270:13010-13016, 1995 and references therein, which are each herebyincorporated by reference in their entireties, particularly with respectto assays for isoprene synthase polypeptide activity. DMAPP (Sigma) isevaporated to dryness under a stream of nitrogen and rehydrated to aconcentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 andstored at −20° C. To perform the assay, a solution of 5 μl of 1M MgCl₂,1 mM (250

g/ml) DMAPP, 65

l of Plant Extract Buffer (PEB) (50 mM Tris-HCl, pH 8.0, 20 mM MgCl₂, 5%glycerol, and 2 mM DTT) is added to 25

l of cell extract in a 20 ml Headspace vial with a metal screw cap andteflon coated silicon septum (Agilent Technologies) and cultured at 37°C. for 15 minutes with shaking. The reaction is quenched by adding 200

l of 250 mM EDTA and quantified by GC/MS as described in Example 1, partII.

Exemplary isoprene synthase nucleic acids include nucleic acids thatencode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an isoprene synthasepolypeptide. Exemplary isoprene synthase polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In some embodiments, the isoprene synthase polypeptide or nucleic acidis from the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the isoprene synthase polypeptide or nucleic acid is apolypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey etal., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (suchas Populus alba, Populus nigra, Populus trichocarpa, Populus alba xtremula (CAC35696), or Populus alba) (Sasaki et al., FEBS Letters579(11): 2514-2518, 2005; Miller et al., Planta 213: 483-487, 2001),aspen (such as Populus tremuloides) (Silver et al., JBC 270(22):13010-1316, 1995), or English Oak (Quercus robur) (Zimmer et al., WO98/02550), which are each hereby incorporated by reference in theirentireties, particularly with respect to isoprene synthase nucleic acidsand the expression of isoprene synthase polypeptides. Suitable isoprenesynthases include, but are not limited to, those identified by GenbankAccession Nos. AY341431, AY316691, AY279379, AJ457070, and AY182241,which are each hereby incorporated by reference in their entireties,particularly with respect to sequences of isoprene synthase nucleicacids and polypeptides. In some embodiments, the isoprene synthasepolypeptide or nucleic acid is not a naturally-occurring polypeptide ornucleic acid from Quercus robur (i.e., the isoprene synthase polypeptideor nucleic acid is an isoprene synthase polypeptide or nucleic acidother than a naturally-occurring polypeptide or nucleic acid fromQuercus robur). In some embodiments, the isoprene synthase nucleic acidor polypeptide is a naturally-occurring polypeptide or nucleic acid frompoplar. In some embodiments, the isoprene synthase nucleic acid orpolypeptide is not a naturally-occurring polypeptide or nucleic acidfrom poplar.

Exemplary DXS Polypeptides and Nucleic Acids

As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS)polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into1-deoxy-D-xylulose-5-phosphate. Exemplary DXS polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of a DXS polypeptide.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has DXS polypeptide activity bymeasuring the ability of the polypeptide to convert pyruvate andD-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate invitro, in a cell extract, or in vivo. Exemplary DXS nucleic acidsinclude nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of a DXS polypeptide. Exemplary DXS polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary IDI Polypeptides and Nucleic Acids

Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphatedelta-isomerase or IDI) catalyses the interconversion of isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (e.g.,converting IPP into DMAPP and/or converting DMAPP into IPP). ExemplaryIDI polypeptides include polypeptides, fragments of polypeptides,peptides, and fusions polypeptides that have at least one activity of anIDI polypeptide. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has IDI polypeptide activityby measuring the ability of the polypeptide to interconvert IPP andDMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleicacids include nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of an IDI polypeptide. Exemplary IDI polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary MVA Pathway Polypeptides and Nucleic Acids

Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase(AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase(HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoAreductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK)polypeptides, phosphomevalonate kinase (PMK) polypeptides,diphosphomevalonte decarboxylase (MVD) polypeptides, phosphomevalonatedecarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK)polypeptides, IDI polypeptides, and polypeptides (e.g., fusionpolypeptides) having an activity of two or more MVA pathwaypolypeptides. In particular, MVA pathway polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway nucleic acids include nucleic acidsthat encode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoAthiolase or AACT) convert two molecules of acetyl-CoA intoacetoacetyl-CoA. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has AA-CoA thiolasepolypeptide activity by measuring the ability of the polypeptide toconvert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in acell extract, or in vivo.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase or HMGS)polypeptides convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA. Standard methods (such as thosedescribed herein) can be used to determine whether a polypeptide hasHMG-CoA synthase polypeptide activity by measuring the ability of thepolypeptide to convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR)polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has HMG-CoA reductase polypeptideactivity by measuring the ability of the polypeptide to convert3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cellextract, or in vivo.

Mevalonate kinase (MVK) polypeptide phosphorylates mevalonate to formmevalonate-5-phosphate. Standard methods (such as those describedherein) can be used to determine whether a polypeptide has MVKpolypeptide activity by measuring the ability of the polypeptide toconvert mevalonate into mevalonate-5-phosphate in vitro, in a cellextract, or in vivo.

Phosphomevalonate kinase (PMK) polypeptides phosphorylatesmevalonate-5-phosphate to form mevalonate-5-diphosphate. Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has PMK polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-phosphate intomevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.

Diphosphomevalonte decarboxylase (MVD or DPMDC) polypeptides convertmevalonate-5-diphosphate into isopentenyl diphosphate (IPP). Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has MVD polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-diphosphate into IPPin vitro, in a cell extract, or in vivo.

Phosphomevalonate decarboxylase (PMDC) polypeptides convertmevalonate-5-phosphate into isopentenyl phosphate (IP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has PMDC polypeptide activity by measuring the ability ofthe polypeptide to convert mevalonate-5-phosphate into IP in vitro, in acell extract, or in vivo.

Isopentenyl phosphate kinase (IPK) polypeptides phosphorylate isopentylphosphate (IP) to form isopentenyl diphosphate (IPP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has IPK polypeptide activity by measuring the ability of thepolypeptide to convert IP into IPP in vitro, in a cell extract, or invivo.

Exemplary IDI polypeptides and nucleic acids are described above.

Exemplary Methods for Isolating Nucleic Acids

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids can beisolated using standard methods. Methods of obtaining desired nucleicacids from a source organism of interest (such as a bacterial genome)are common and well known in the art of molecular biology (see, forexample, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to the isolation of nucleic acids of interest). For example, ifthe sequence of the nucleic acid is known (such as any of the knownnucleic acids described herein), suitable genomic libraries may becreated by restriction endonuclease digestion and may be screened withprobes complementary to the desired nucleic acid sequence. Once thesequence is isolated, the DNA may be amplified using standard primerdirected amplification methods such as polymerase chain reaction (PCR)(U.S. Pat. No. 4,683,202, which is incorporated by reference in itsentirety, particularly with respect to PCR methods) to obtain amounts ofDNA suitable for transformation using appropriate vectors.

Alternatively, isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacids (such as any isoprene synthase, DXS, IDI, and/or MVA pathwaynucleic acids with a known nucleic acid sequence) can be chemicallysynthesized using standard methods.

Additional isoprene synthase, DXS, IDI, or MVA pathway polypeptides andnucleic acids which may be suitable for use in the compositions andmethods described herein can be identified using standard methods. Forexample, cosmid libraries of the chromosomal DNA of organisms known toproduce isoprene naturally can be constructed in organisms such as E.coli, and then screened for isoprene production. In particular, cosmidlibraries may be created where large segments of genomic DNA (35-45 kb)are packaged into vectors and used to transform appropriate hosts.Cosmid vectors are unique in being able to accommodate large quantitiesof DNA. Generally cosmid vectors have at least one copy of the cos DNAsequence which is needed for packaging and subsequent circularization ofthe heterologous DNA. In addition to the cos sequence, these vectorsalso contain an origin of replication such as ColEI and drug resistancemarkers such as a nucleic acid resistant to ampicillin or neomycin.Methods of using cosmid vectors for the transformation of suitablebacterial hosts are well described in Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989,which is hereby incorporated by reference in its entirety, particularlywith respect to transformation methods.

Typically to clone cosmids, heterologous DNA is isolated using theappropriate restriction endonucleases and ligated adjacent to the cosregion of the cosmid vector using the appropriate ligases. Cosmidvectors containing the linearized heterologous DNA are then reacted witha DNA packaging vehicle such as bacteriophage. During the packagingprocess, the cos sites are cleaved and the heterologous DNA is packagedinto the head portion of the bacterial viral particle. These particlesare then used to transfect suitable host cells such as E. coli. Onceinjected into the cell, the heterologous DNA circularizes under theinfluence of the cos sticky ends. In this manner, large segments ofheterologous DNA can be introduced and expressed in host cells.

Additional methods for obtaining isoprene synthase, DXS, IDI, and/or MVApathway nucleic acids include screening a metagenomic library by assay(such as the headspace assay described herein) or by PCR using primersdirected against nucleotides encoding for a length of conserved aminoacids (for example, at least 3 conserved amino acids). Conserved aminoacids can be identified by aligning amino acid sequences of knownisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides. Conservedamino acids for isoprene synthase polypeptides can be identified basedon aligned sequences of known isoprene synthase polypeptides. Anorganism found to produce isoprene naturally can be subjected tostandard protein purification methods (which are well known in the art)and the resulting purified polypeptide can be sequenced using standardmethods. Other methods are found in the literature (see, for example,Julsing et al., Applied. Microbiol. Biotechnol. 75: 1377-84, 2007;Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which areeach hereby incorporated by reference in their entireties, particularlywith respect to identification of nucleic acids involved in thesynthesis of isoprene).

Additionally, standard sequence alignment and/or structure predictionprograms can be used to identify additional DXS, IDI, or MVA pathwaypolypeptides and nucleic acids based on the similarity of their primaryand/or predicted polypeptide secondary structure with that of known DXS,IDI, or MVA pathway polypeptides and nucleic acids. Standard databasessuch as the swissprot-trembl database (world-wide web at “expasy.org”,Swiss Institute of Bioinformatics Swiss-Prot group CMU—1 rue MichelServet CH-1211 Geneva 4, Switzerland) can also be used to identifyisoprene synthase, DXS, IDI, or MVA pathway polypeptides and nucleicacids. The secondary and/or tertiary structure of an isoprene synthase,DXS, IDI, or MVA pathway polypeptide can be predicted using the defaultsettings of standard structure prediction programs, such asPredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA).Alternatively, the actual secondary and/or tertiary structure of anisoprene synthase, DXS, IDI, or MVA pathway polypeptide can bedetermined using standard methods. Additional isoprene synthase, DXS,IDI, or MVA pathway nucleic acids can also be identified byhybridization to probes generated from known isoprene synthase, DXS,IDI, or MVA pathway nucleic acids.

Exemplary Promoters and Vectors

Any of the isoprene synthase, DXS, IDI, or MVA pathway nucleic aciddescribed herein can be included in one or more vectors. Accordingly,the invention also features vectors with one more nucleic acids encodingany of the isoprene synthase, DXS, IDI, or MVA pathway polypeptides thatare described herein. As used herein, a “vector” means a construct thatis capable of delivering, and desirably expressing one or more nucleicacids of interest in a host cell. Examples of vectors include, but arenot limited to, plasmids, viral vectors, DNA or RNA expression vectors,cosmids, and phage vectors. In some embodiments, the vector contains anucleic acid under the control of an expression control sequence.

As used herein, an “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid of interest. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. An “inducible promoter” is apromoter that is active under environmental or developmental regulation.The expression control sequence is operably linked to the nucleic acidsegment to be transcribed.

In some embodiments, the vector contains a selective marker. The term“selective marker” refers to a nucleic acid capable of expression in ahost cell that allows for ease of selection of those host cellscontaining an introduced nucleic acid or vector. Examples of selectablemarkers include, but are not limited to, antibiotic resistance nucleicacids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin,hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/ornucleic acids that confer a metabolic advantage, such as a nutritionaladvantage on the host cell. Exemplary nutritional selective markersinclude those markers known in the art as amdS, argB, and pyr4. Markersuseful in vector systems for transformation of Trichoderma are known inthe art (see, e.g., Finkelstein, Chapter 6 in Biotechnology ofFilamentous Fungi, Finkelstein et al., Eds. Butterworth-Heinemann,Boston, Mass., Chap. 6., 1992; and Kinghorn et al., Applied MolecularGenetics of Filamentous Fungi, Blackie Academic and Professional,Chapman and Hall, London, 1992, which are each hereby incorporated byreference in their entireties, particularly with respect to selectivemarkers). In some embodiments, the selective marker is the amdS nucleicacid, which encodes the enzyme acetamidase, allowing transformed cellsto grow on acetamide as a nitrogen source. The use of an A. nidulansamdS nucleic acid as a selective marker is described in Kelley et al.,EMBO J. 4:475-479, 1985 and Penttila et al., Gene 61:155-164, 1987(which are each hereby incorporated by reference in their entireties,particularly with respect to selective markers). In some embodiments, anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid integrates intoa chromosome of the cells without a selective marker.

Suitable vectors are those which are compatible with the host cellemployed. Suitable vectors can be derived, for example, from abacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), acosmid, a yeast, or a plant. Protocols for obtaining and using suchvectors are known to those in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor, 1989, which is hereby incorporated by reference in its entirety,particularly with respect to the use of vectors).

Promoters are well known in the art. Any promoter that functions in thehost cell can be used for expression of an isoprene synthase, DXS, IDI,or MVA pathway nucleic acid in the host cell. Initiation control regionsor promoters, which are useful to drive expression of isoprene synthase,DXS, IDI, or MVA pathway nucleic acids in various host cells arenumerous and familiar to those skilled in the art (see, for example, WO2004/033646 and references cited therein, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors for the expression of nucleic acids of interest). Virtuallyany promoter capable of driving these nucleic acids is suitable for thepresent invention including, but not limited to, CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI(useful for expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, trp,

P_(L),

P_(R), T7, tac, and trc (useful for expression in E. coli).

In some embodiments, a glucose isomerase promoter is used (see, forexample, U.S. Pat. No. 7,132,527 and references cited therein, which areeach hereby incorporated by reference in their entireties, particularlywith respect promoters and plasmid systems for expressing polypeptidesof interest). Reported glucose isomerase promoter mutants can be used tovary the level of expression of the polypeptide encoded by a nucleicacid operably linked to the glucose isomerase promoter (U.S. Pat. No.7,132,527). In various embodiments, the glucose isomerase promoter iscontained in a low, medium, or high copy plasmid (U.S. Pat. No.7,132,527).

In various embodiments, an isoprene synthase, DXS, IDI, and/or MVApathway nucleic acid is contained in a low copy plasmid (e.g., a plasmidthat is maintained at about 1 to about 4 copies per cell), medium copyplasmid (e.g., a plasmid that is maintained at about 10 to about 15copies per cell), or high copy plasmid (e.g., a plasmid that ismaintained at about 50 or more copies per cell). In some embodiments,the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a T7 promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a T7 promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Trc promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Trc promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Lac promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Lac promoteris contained in a low copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to an endogenous promoter, suchas an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces,or Trichoderma promoter or an endogenous alkaline serine protease,isoprene synthase, DXS, IDI, or MVA pathway promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to an endogenouspromoter is contained in a high copy plasmid. In some embodiments, thevector is a replicating plasmid that does not integrate into achromosome in the cells. In some embodiments, part or all of the vectorintegrates into a chromosome in the cells.

In some embodiments, the vector is any vector which when introduced intoa fungal host cell is integrated into the host cell genome and isreplicated. Reference is made to the Fungal Genetics Stock CenterCatalogue of Strains (FGSC, the world-wide web at “fgsc.net” and thereferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect to vectors) fora list of vectors. Additional examples of suitable expression and/orintegration vectors are provided in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, CurrentProtocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987,Supplement 30, section 7.7.18); van den Hondel et al. in Bennett andLasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp.396-428, 1991; and U.S. Pat. No. 5,874,276, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors. Particularly useful vectors include pFB6, pBR322, PUC18,pUC100, and pENTR/D.

In some embodiments, an isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid is operably linked to a suitable promoter that showstranscriptional activity in a fungal host cell. The promoter may bederived from one or more nucleic acids encoding a polypeptide that iseither endogenous or heterologous to the host cell. In some embodiments,the promoter is useful in a Trichoderma host. Suitable non-limitingexamples of promoters include cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2,xyn1, and amy. In some embodiments, the promoter is one that is nativeto the host cell. For example, in some embodiments when T. reesei is thehost, the promoter is a native T. reesei promoter. In some embodiments,the promoter is T. reesei cbh1, which is an inducible promoter and hasbeen deposited in GenBank under Accession No. D86235, which isincorporated by reference in its entirety, particularly with respect topromoters. In some embodiments, the promoter is one that is heterologousto the fungal host cell. Other examples of useful promoters includepromoters from the genes of A. awamori and A. niger glucoamylase (glaA)(Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel et al., EMBOJ. 3:1581-1585, 1984, which are each hereby incorporated by reference intheir entireties, particularly with respect to promoters); Aspergillusniger alpha amylases, Aspergillus oryzae TAKA amylase, T. reesei xln1,and the T. reesei cellobiohydrolase 1 (EP 137280, which is incorporatedby reference in its entirety, particularly with respect to promoters).

In some embodiments, the expression vector also includes a terminationsequence. Termination control regions may also be derived from variousgenes native to the host cell. In some embodiments, the terminationsequence and the promoter sequence are derived from the same source. Inanother embodiment, the termination sequence is endogenous to the hostcell. A particularly suitable terminator sequence is cbh1 derived from aTrichoderma strain (such as T. reesei). Other useful fungal terminatorsinclude the terminator from an A. niger or A. awamori glucoamylasenucleic acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boelet al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated byreference in their entireties, particularly with respect to fungalterminators). Optionally, a termination site may be included. Foreffective expression of the polypeptides, DNA encoding the polypeptideare linked operably through initiation codons to selected expressioncontrol regions such that expression results in the formation of theappropriate messenger RNA.

In some embodiments, the promoter, coding, region, and terminator alloriginate from the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid to be expressed. In some embodiments, the coding region for anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid is insertedinto a general-purpose expression vector such that it is under thetranscriptional control of the expression construct promoter andterminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

An isoprene synthase, DXS, IDI, or MVA pathway nucleic acid can beincorporated into a vector, such as an expression vector, using standardtechniques (Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, 1982, which is hereby incorporated by reference inits entirety, particularly with respect to the screening of appropriateDNA sequences and the construction of vectors). Methods used to ligatethe DNA construct comprising a nucleic acid of interest (such as anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid), a promoter, aterminator, and other sequences and to insert them into a suitablevector are well known in the art. For example, restriction enzymes canbe used to cleave the isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid and the vector. Then, the compatible ends of the cleavedisoprene synthase, DXS, IDI, or MVA pathway nucleic acid and the cleavedvector can be ligated. Linking is generally accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide linkers are used in accordance with conventionalpractice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More GeneManipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to oligonucleotide linkers). Additionally,vectors can be constructed using known recombination techniques (e.g.,Invitrogen Life Technologies, Gateway Technology).

In some embodiments, it may be desirable to over-express isoprenesynthase, DXS, IDI, or MVA pathway nucleic acids at levels far higherthan currently found in naturally-occurring cells. This result may beaccomplished by the selective cloning of the nucleic acids encodingthose polypeptides into multicopy plasmids or placing those nucleicacids under a strong inducible or constitutive promoter. Methods forover-expressing desired polypeptides are common and well known in theart of molecular biology and examples may be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor,1989, which is hereby incorporated by reference in its entirety,particularly with respect to cloning techniques.

The following resources include descriptions of additional generalmethodology useful in accordance with the invention: Kreigler, GeneTransfer and Expression; A Laboratory Manual, 1990 and Ausubel et al.,Eds. Current Protocols in Molecular Biology, 1994, which are each herebyincorporated by reference in their entireties, particularly with respectto molecular biology and cloning techniques.

Exemplary Source Organisms

Isoprene synthase, DXS, IDI, or MVA pathway nucleic acids (and theirencoded polypeptides) can be obtained from any organism that naturallycontains isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids.As noted above, isoprene is formed naturally by a variety of organisms,such as bacteria, yeast, plants, and animals. Organisms contain the MVApathway, DXP pathway, or both the MVA and DXP pathways for producingisoprene (FIG. 19). Thus, DXS nucleic acids can be obtained, e.g., fromany organism that contains the DXP pathway or contains both the MVA andDXP pathways. IDI and isoprene synthase nucleic acids can be obtained,e.g., from any organism that contains the MVA pathway, DXP pathway, orboth the MVA and DXP pathways. MVA pathway nucleic acids can beobtained, e.g., from any organism that contains the MVA pathway orcontains both the MVA and DXP pathways.

In some embodiments, the nucleic acid sequence of the isoprene synthase,DXS, IDI, or MVA pathway nucleic is identical to the sequence of anucleic acid that is produced by any of the following organisms innature. In some embodiments, the amino acid sequence of the isoprenesynthase, DXS, IDI, or MVA pathway polypeptide is identical to thesequence of a polypeptide that is produced by any of the followingorganisms in nature. In some embodiments, the isoprene synthase, DXS,IDI, or MVA pathway nucleic acid or polypeptide is a mutant nucleic acidor polypeptide derived from any of the organisms described herein. Asused herein, “derived from” refers to the source of the nucleic acid orpolypeptide into which one or more mutations is introduced. For example,a polypeptide that is “derived from a plant polypeptide” refers topolypeptide of interest that results from introducing one or moremutations into the sequence of a wild-type (i.e., a sequence occurringin nature) plant polypeptide.

In some embodiments, the source organism is a fungus, examples of whichare species of Aspergillus such as A. oryzae and A. niger, species ofSaccharomyces such as S. cerevisiae, species of Schizosaccharomyces suchas S. pombe, and species of Trichoderma such as T. reesei. In someembodiments, the source organism is a filamentous fungal cell. The term“filamentous fungi” refers to all filamentous forms of the subdivisionEumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology,Wiley, New York). These fungi are characterized by a vegetative myceliumwith a cell wall composed of chitin, cellulose, and other complexpolysaccharides. The filamentous fungi are morphologically,physiologically, and genetically distinct from yeasts. Vegetative growthby filamentous fungi is by hyphal elongation and carbon catabolism isobligatory aerobic. The filamentous fungal parent cell may be a cell ofa species of, but not limited to, Trichoderma, (e.g., Trichodermareesei, the asexual morph of Hypocrea jecorina, previously classified asT. longibrachiatum, Trichoderma viride, Trichoderma koningii,Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp.,Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea);Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillussp. (e.g., A. oryzae, A. niger, A sojae, A. japonicus, A. nidulans, orA. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993and Goedegebuur et al., Genet 41: 89-98, 2002), Fusarium sp., (e.g., F.roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum),Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M.miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci.228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or“Trichoderma spp.” refer to any fungal genus previously or currentlyclassified as Trichoderma.

In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae,A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F.oxysporum, or F. solani. Aspergillus strains are disclosed in Ward etal., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur etal., Curr Gene 41:89-98, 2002, which are each hereby incorporated byreference in their entireties, particularly with respect to fungi. Inparticular embodiments, the fungus is a strain of Trichoderma, such as astrain of T. reesei. Strains of T. reesei are known and non-limitingexamples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCCNo. 56765, ATCC No. 56767, and NRRL 15709, which are each herebyincorporated by reference in their entireties, particularly with respectto strains of T. reesei. In some embodiments, the host strain is aderivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl.Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated byreference in its entirety, particularly with respect to strains of T.reesei.

In some embodiments, the source organism is a yeast, such asSaccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.

In some embodiments, the source organism is a bacterium, such as strainsof Bacillus such as B. lichenformis or B. subtilis, strains of Pantoeasuch as P. citrea, strains of Pseudomonas such as P. alcaligenes,strains of Streptomyces such as S. albus, S. lividans, or S.rubiginosus, or strains of Escherichia such as E. coli.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

In some embodiments, the source organism is a gram-positive bacterium.Non-limiting examples include strains of Streptomyces (e.g., S. albus,S. lividans, S. coelicolor, or S. griseus) and Bacillus. In someembodiments, the source organism is a gram-negative bacterium, such asE. coli or Pseudomonas sp.

In some embodiments, the source organism is a plant, such as a plantfrom the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the source organism is kudzu, poplar (such as Populus albax tremula CAC35696 or Populus alba) (Sasaki et al., FEBS Letters579(11): 2514-2518, 2005), aspen (such as Populus tremuloides), orQuercus robur.

In some embodiments, the source organism is an algae, such as a greenalgae, red algae, glaucophytes, chlorarachniophytes, euglenids,chromista, or dinoflagellates.

In some embodiments, the source organism is a cyanobacteria, such ascyanobacteria classified into any of the following groups based onmorphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales,or Stigonematales.

In some embodiments, the source organism is an anaerobic organism.Anaerobic organisms can include, but are not limited to, obligateanaerobes, facultative anaerobes, and aerotolerant anaerobes. Suchorganisms can be any of the organisms listed above, bacteria, yeast,etc. In one embodiment, the obligate anaerobes can be any one orcombination selected from the group consisting of Clostridiumljungdahlii, Clostridium autoethanogenum, Eurobacterium limosum,Clostridium carboxydivorans, Peptostreptococcus productus, andButyribacterium methylotrophicum. It is to be understood that anycombination of any of the source organisms described herein can be usedfor other embodiments of the invention.

Exemplary Host Cells

A variety of host cells can be used to express isoprene synthase, DXS,IDI, and/or MVA pathway polypeptides and to produce isoprene in themethods of the claimed invention. Exemplary host cells include cellsfrom any of the organisms listed in the prior section under the heading“Exemplary Source Organisms.” The host cell may be a cell that naturallyproduces isoprene or a cell that does not naturally produce isoprene. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway, and an isoprene synthase, DXS, and/or IDI nucleic acid isadded to enhance production of isoprene using this pathway. In someembodiments, the host cell naturally produces isoprene using the MVApathway, and an isoprene synthase and/or one or more MVA pathway nucleicacids are added to enhance production of isoprene using this pathway. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway and one or more MVA pathway nucleic acids are added toproduce isoprene using part or all of the MVA pathway as well as the DXPpathway. In some embodiments, the host cell naturally produces isopreneusing both the DXP and MVA pathways and one or more isoprene synthase,DXS, IDI, or MVA pathway nucleic acids are added to enhance productionof isoprene by one or both of these pathways. It is to be understoodthat any combination of any of the host organisms described herein canbe used for other embodiments of the invention.

Exemplary Transformation Methods

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids or vectorscontaining them can be inserted into a host cell (e.g., a plant cell, afungal cell, a yeast cell, or a bacterial cell described herein) usingstandard techniques for expression of the encoded isoprene synthase,DXS, IDI, and/or MVA pathway polypeptide. Introduction of a DNAconstruct or vector into a host cell can be performed using techniquessuch as transformation, electroporation, nuclear microinjection,transduction, transfection (e.g., lipofection mediated or DEAE-Dextrinmediated transfection or transfection using a recombinant phage virus),incubation with calcium phosphate DNA precipitate, high velocitybombardment with DNA-coated microprojectiles, and protoplast fusion.General transformation techniques are known in the art (see, e.g.,Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds)Chapter 9, 1987; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor, 1989; and Campbell et al., Curr.Genet. 16:53-56, 1989, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods). The expression of heterologous polypeptide in Trichoderma isdescribed in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; U.S. Pat.No. 7,262,041; WO 2005/001036; Harkki et al.; Enzyme Microb. Technol.13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology ofTrichoderma and its Application to the Expression of Both Homologous andHeterologous Genes,” in Molecular Industrial Mycology, Eds. Leong andBerka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each herebyincorporated by reference in their entireties, particularly with respectto transformation and expression methods). Reference is also made to Caoet al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al.,Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are eachhereby incorporated by reference in their entireties, particularly withrespect to transformation methods) for transformation of Aspergillusstrains. The introduced nucleic acids may be integrated into chromosomalDNA or maintained as extrachromosomal replicating sequences.

Any method known in the art may be used to select transformants. In onenon-limiting example, stable transformants including an amdS marker aredistinguished from unstable transformants by their faster growth rateand the formation of circular colonies with a smooth, rather than raggedoutline on solid culture medium containing acetamide. Additionally, insome cases a further test of stability is conducted by growing thetransformants on a solid non-selective medium (e.g., a medium that lacksacetamide), harvesting spores from this culture medium, and determiningthe percentage of these spores which subsequently germinate and grow onselective medium containing acetamide.

In some embodiments, fungal cells are transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a known manner. In one specificembodiment, the preparation of Trichoderma sp. for transformationinvolves the preparation of protoplasts from fungal mycelia (see,Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated byreference in its entirety, particularly with respect to transformationmethods). In some embodiments, the mycelia are obtained from germinatedvegetative spores. The mycelia are treated with an enzyme that digeststhe cell wall resulting in protoplasts. The protoplasts are thenprotected by the presence of an osmotic stabilizer in the suspendingmedium. These stabilizers include sorbitol, mannitol, potassiumchloride, magnesium sulfate, and the like. Usually the concentration ofthese stabilizers varies between 0.8 M and 1.2 M. It is desirable to useabout a 1.2 M solution of sorbitol in the suspension medium.

Uptake of DNA into the host Trichoderma sp. strain is dependent upon thecalcium ion concentration. Generally, between about 10 mM CaCl₂ and 50mM CaCl₂ is used in an uptake solution. In addition to the calcium ionin the uptake solution, other compounds generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). While not intending to be bound to any particular theory,it is believed that the polyethylene glycol acts to fuse the cellmembranes, thus permitting the contents of the medium to be deliveredinto the cytoplasm of the Trichoderma sp. strain and the plasmid DNA tobe transferred to the nucleus. This fusion frequently leaves multiplecopies of the plasmid DNA integrated into the host chromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁵to 10⁷/mL (such as 2×10⁶/mL) are used in the transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol and 50 mM CaCl₂) are mixed with the desired DNA.Generally, a high concentration of PEG is added to the uptake solution.From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplastsuspension. In some embodiments, about 0.25 volumes are added to theprotoplast suspension. Additives such as dimethyl sulfoxide, heparin,spermidine, potassium chloride, and the like may also be added to theuptake solution and aid in transformation. Similar procedures areavailable for other fungal host cells (see, e.g., U.S. Pat. No.6,022,725 and U.S. Pat. No. 6,268,328, which are each herebyincorporated by reference in their entireties, particularly with respectto transformation methods).

Generally, the mixture is then cultured at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired nucleic acidsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is desirably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then cultured either at room temperatureor on ice before the addition of a sorbitol and CaCl₂ solution. Theprotoplast suspension is then further added to molten aliquots of agrowth medium. When the growth medium includes a growth selection (e.g.,acetamide or an antibiotic) it permits the growth of transformants only.

The transformation of bacterial cells may be performed according toconventional methods, e.g., as described in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is herebyincorporated by reference in its entirety, particularly with respect totransformation methods.

Exemplary Cell Culture Media

The invention also includes a cell or a population of cells in culturethat produce isoprene. By “cells in culture” is meant two or more cellsin a solution (e.g., a cell medium) that allows the cells to undergo oneor more cell divisions. “Cells in culture” do not include plant cellsthat are part of a living, multicellular plant containing cells thathave differentiated into plant tissues. In various embodiments, the cellculture includes at least or about 10, 20, 50, 100, 200, 500, 1,000,5,000, 10,000 or more cells.

Any carbon source can be used to cultivate the host cells. The term“carbon source” refers to one or more carbon-containing compoundscapable of being metabolized by a host cell or organism. For example,the cell medium used to cultivate the host cells may include any carbonsource suitable for maintaining the viability or growing the host cells.

In some embodiments, the carbon source is a carbohydrate (such asmonosaccharide, disaccharide, oligosaccharide, or polysaccharids),invert sugar (e.g., enzymatically treated sucrose syrup), glycerol,glycerine (e.g., a glycerine byproduct of a biodiesel or soap-makingprocess), dihydroxyacetone, one-carbon source, oil (e.g., a plant orvegetable oil such as corn, palm, or soybean oil), acetate, animal fat,animal oil, fatty acid (e.g., a saturated fatty acid, unsaturated fattyacid, or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbialor plant protein or peptide), renewable carbon source (e.g., a biomasscarbon source such as a hydrolyzed biomass carbon source), yeastextract, component from a yeast extract, polymer, acid, alcohol,aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, orany combination of two or more of the foregoing. In some embodiments,the carbon source is a product of photosynthesis, including, but notlimited to, glucose. In some embodiment, the carbohydrate is xylose orglucose.

Exemplary monosaccharides include glucose and fructose; exemplaryoligosaccharides include lactose and sucrose, and exemplarypolysaccharides include starch and cellulose. Exemplary carbohydratesinclude C6 sugars (e.g., fructose, mannose, galactose, or glucose) andC5 sugars (e.g., xylose or arabinose). In some embodiments, the cellmedium includes a carbohydrate as well as a carbon source other than acarbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, or a component from a yeast extract). In some embodiments, thecell medium includes a carbohydrate as well as a polypeptide (e.g., amicrobial or plant protein or peptide). In some embodiments, themicrobial polypeptide is a polypeptide from yeast or bacteria. In someembodiments, the plant polypeptide is a polypeptide from soy, corn,canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In some embodiments, the concentration of the carbohydrate is at leastor about 5 grams per liter of broth (g/L, wherein the volume of brothincludes both the volume of the cell medium and the volume of thecells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100,150, 200, 300, 400, or more g/L. In some embodiments, the concentrationof the carbohydrate is between about 50 and about 400 g/L, such asbetween about 100 and about 360 g/L, between about 120 and about 360g/L, or between about 200 and about 300 g/L. In some embodiments, thisconcentration of carbohydrate includes the total amount of carbohydratethat is added before and/or during the culturing of the host cells.

In some embodiments, the cells are cultured under limited glucoseconditions. By “limited glucose conditions” is meant that the amount ofglucose that is added is less than or about 105% (such as about 100%) ofthe amount of glucose that is consumed by the cells. In particularembodiments, the amount of glucose that is added to the culture mediumis approximately the same as the amount of glucose that is consumed bythe cells during a specific period of time. In some embodiments, therate of cell growth is controlled by limiting the amount of addedglucose such that the cells grow at the rate that can be supported bythe amount of glucose in the cell medium. In some embodiments, glucosedoes not accumulate during the time the cells are cultured. In variousembodiments, the cells are cultured under limited glucose conditions forgreater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or70 hours. In various embodiments, the cells are cultured under limitedglucose conditions for greater than or about 5, 10, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time thecells are cultured. While not intending to be bound by any particulartheory, it is believed that limited glucose conditions may allow morefavorable regulation of the cells.

In some embodiments, the cells are cultured in the presence of an excessof glucose. In particular embodiments, the amount of glucose that isadded is greater than about 105% (such as about or greater than 110,120, 150, 175, 200, 250, 300, 400, or 500%) or more of the amount ofglucose that is consumed by the cells during a specific period of time.In some embodiments, glucose accumulates during the time the cells arecultured.

Exemplary lipids are any substance containing one or more fatty acidsthat are C4 and above fatty acids that are saturated, unsaturated, orbranched.

Exemplary oils are lipids that are liquid at room temperature. In someembodiments, the lipid contains one or more C4 or above fatty acids(e.g., contains one or more saturated, unsaturated, or branched fattyacid with four or more carbons). In some embodiments, the oil isobtained from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, linseed, oleagineous microbial cells, Chinese tallow, or anycombination of two or more of the foregoing.

Exemplary fatty acids include compounds of the formula RCOOH, where “R”is a hydrocarbon. Exemplary unsaturated fatty acids include compoundswhere “R” includes at least one carbon-carbon double bond. Exemplaryunsaturated fatty acids include, but are not limited to, oleic acid,vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid.Exemplary polyunsaturated fatty acids include compounds where “R”includes a plurality of carbon-carbon double bonds. Exemplary saturatedfatty acids include compounds where “R” is a saturated aliphatic group.In some embodiments, the carbon source includes one or more C₁₂-C₂₂fatty acids, such as a C₁₂ saturated fatty acid, a C₁₄ saturated fattyacid, a C₁₆ saturated fatty acid, a C₁₈ saturated fatty acid, a C₂₀saturated fatty acid, or a C₂₂ saturated fatty acid. In an exemplaryembodiment, the fatty acid is palmitic acid. In some embodiments, thecarbon source is a salt of a fatty acid (e.g., an unsaturated fattyacid), a derivative of a fatty acid (e.g., an unsaturated fatty acid),or a salt of a derivative of fatty acid (e.g., an unsaturated fattyacid). Suitable salts include, but are not limited to, lithium salts,potassium salts, sodium salts, and the like. Di- and triglycerols arefatty acid esters of glycerol.

In some embodiments, the concentration of the lipid, oil, fat, fattyacid, monoglyceride, diglyceride, or triglyceride is at least or about 1gram per liter of broth (g/L, wherein the volume of broth includes boththe volume of the cell medium and the volume of the cells), such as atleast or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300,400, or more g/L. In some embodiments, the concentration of the lipid,oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride isbetween about 10 and about 400 g/L, such as between about 25 and about300 g/L, between about 60 and about 180 g/L, or between about 75 andabout 150 g/L. In some embodiments, the concentration includes the totalamount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride,or triglyceride that is added before and/or during the culturing of thehost cells. In some embodiments, the carbon source includes both (i) alipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglycerideand (ii) a carbohydrate, such as glucose. In some embodiments, the ratioof the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, ortriglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e.,one carbon in the lipid, oil, fat, fatty acid, monoglyceride,diglyceride, or triglyceride per carbohydrate carbon). In particularembodiments, the amount of the lipid, oil, fat, fatty acid,monoglyceride, diglyceride, or triglyceride is between about 60 and 180g/L, and the amount of the carbohydrate is between about 120 and 360g/L.

Exemplary microbial polypeptide carbon sources include one or morepolypeptides from yeast or bacteria. Exemplary plant polypeptide carbonsources include one or more polypeptides from soy, corn, canola,jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Exemplary renewable carbon sources include cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt, and components fromany of the foregoing. Exemplary renewable carbon sources also includeacetate, glucose, hexose, pentose and xylose present in biomass, such ascorn, switchgrass, sugar cane, cell waste of fermentation processes, andprotein by-product from the milling of soy, corn, or wheat. In someembodiments, the biomass carbon source is a lignocellulosic,hemicellulosic, or cellulosic material such as, but are not limited to,a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft woodpulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, cornstover, switch grass, rice hull product, or a by-product from wet or drymilling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat,and/or distillers grains). Exemplary cellulosic materials include wood,paper and pulp waste, herbaceous plants, and fruit pulp. In someembodiments, the carbon source includes any plant part, such as stems,grains, roots, or tubers. In some embodiments, all or part of any of thefollowing plants are used as a carbon source: corn, wheat, rye, sorghum,triticate, rice, millet, barley, cassava, legumes, such as beans andpeas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. Insome embodiments, the carbon source is a biomass hydrolysate, such as abiomass hydrolysate that includes both xylose and glucose or thatincludes both sucrose and glucose.

In some embodiments, the renewable carbon source (such as biomass) ispretreated before it is added to the cell culture medium. In someembodiments, the pretreatment includes enzymatic pretreatment, chemicalpretreatment, or a combination of both enzymatic and chemicalpretreatment (see, for example, Farzaneh et al., Bioresource Technology96 (18): 2014-2018, 2005; U.S. Pat. No. 6,176,176; U.S. Pat. No.6,106,888; which are each hereby incorporated by reference in theirentireties, particularly with respect to the pretreatment of renewablecarbon sources). In some embodiments, the renewable carbon source ispartially or completely hydrolyzed before it is added to the cellculture medium.

In some embodiments, the renewable carbon source (such as corn stover)undergoes ammonia fiber expansion (AFEX) pretreatment before it is addedto the cell culture medium (see, for example, Farzaneh et al.,Bioresource Technology 96 (18): 2014-2018, 2005). During AFEXpretreatment, a renewable carbon source is treated with liquid anhydrousammonia at moderate temperatures (such as about 60 to about 100° C.) andhigh pressure (such as about 250 to about 300 psi) for about 5 minutes.Then, the pressure is rapidly released. In this process, the combinedchemical and physical effects of lignin solubilization, hemicellulosehydrolysis, cellulose decrystallization, and increased surface areaenables near complete enzymatic conversion of cellulose andhemicellulose to fermentable sugars. AFEX pretreatment has the advantagethat nearly all of the ammonia can be recovered and reused, while theremaining serves as nitrogen source for microbes in downstreamprocesses. Also, a wash stream is not required for AFEX pretreatment.Thus, dry matter recovery following the AFEX treatment is essentially100%. AFEX is basically a dry to dry process. The treated renewablecarbon source is stable for long periods and can be fed at very highsolid loadings in enzymatic hydrolysis or fermentation processes.Cellulose and hemicellulose are well preserved in the AFEX process, withlittle or no degradation. There is no need for neutralization prior tothe enzymatic hydrolysis of a renewable carbon source that has undergoneAFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sourcesproduces clean sugar streams for subsequent fermentation use.

In some embodiments, the concentration of the carbon source (e.g., arenewable carbon source) is equivalent to at least or about 0.1, 0.5, 1,1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalentamount of glucose can be determined by using standard HPLC methods withglucose as a reference to measure the amount of glucose generated fromthe carbon source. In some embodiments, the concentration of the carbonsource (e.g., a renewable carbon source) is equivalent to between about0.1 and about 20% glucose, such as between about 0.1 and about 10%glucose, between about 0.5 and about 10% glucose, between about 1 andabout 10% glucose, between about 1 and about 5% glucose, or betweenabout 1 and about 2% glucose.

In some embodiments, the carbon source includes yeast extract or one ormore components of yeast extract. In some embodiments, the concentrationof yeast extract is at least 1 gram of yeast extract per liter of broth(g/L, wherein the volume of broth includes both the volume of the cellmedium and the volume of the cells), such at least or about 5, 10, 15,20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In someembodiments, the concentration of yeast extract is between about 1 andabout 300 g/L, such as between about 1 and about 200 g/L, between about5 and about 200 g/L, between about 5 and about 100 g/L, or between about5 and about 60 g/L. In some embodiments, the concentration includes thetotal amount of yeast extract that is added before and/or during theculturing of the host cells. In some embodiments, the carbon sourceincludes both yeast extract (or one or more components thereof) andanother carbon source, such as glucose. In some embodiments, the ratioof yeast extract to the other carbon source is about 1:5, about 1:10, orabout 1:20 (w/w).

Additionally the carbon source may also be one-carbon substrates such ascarbon dioxide, or methanol. Glycerol production from single carbonsources (e.g., methanol, formaldehyde, or formate) has been reported inmethylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543,1989, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources) and in bacteria (Hunter et.al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).These organisms can assimilate single carbon compounds, ranging inoxidation state from methane to formate, and produce glycerol. Thepathway of carbon assimilation can be through ribulose monophosphate,through serine, or through xylulose-momophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York, 1986, which ishereby incorporated by reference in its entirety, particularly withrespect to carbon sources). The ribulose monophosphate pathway involvesthe condensation of formate with ribulose-5-phosphate to form a sixcarbon sugar that becomes fructose and eventually the three carbonproduct glyceraldehyde-3-phosphate. Likewise, the serine pathwayassimilates the one-carbon compound into the glycolytic pathway viamethylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organismsare also known to utilize a number of other carbon containing compoundssuch as methylamine, glucosamine and a variety of amino acids formetabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7^(th) ed.,415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK,1993, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources). Similarly, various speciesof Candida metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153(5), 485-9, 1990, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).

In some embodiments, cells are cultured in a standard medium containingphysiological salts and nutrients (see, e.g., Pourquie, J. et al.,Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al.,Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ.Microbiol. 63:1298-1306, 1997, which are each hereby incorporated byreference in their entireties, particularly with respect to cell media).Exemplary growth media are common commercially prepared media such asLuria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium(YM) broth. Other defined or synthetic growth media may also be used,and the appropriate medium for growth of particular host cells are knownby someone skilled in the art of microbiology or fermentation science.

In addition to an appropriate carbon source, the cell medium desirablycontains suitable minerals, salts, cofactors, buffers, and othercomponents known to those skilled in the art suitable for the growth ofthe cultures or the enhancement of isoprene production (see, forexample, WO 2004/033646 and references cited therein and WO 96/35796 andreferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect cell medias andcell culture conditions). In some embodiments where an isoprenesynthase, DXS, IDI, and/or MVA pathway nucleic acid is under the controlof an inducible promoter, the inducing agent (e.g., a sugar, metal saltor antimicrobial), is desirably added to the medium at a concentrationeffective to induce expression of an isoprene synthase, DXS, IDI, and/orMVA pathway polypeptide. In some embodiments, cell medium has anantibiotic (such as kanamycin) that corresponds to the antibioticresistance nucleic acid (such as a kanamycin resistance nucleic acid) ona vector that has one or more DXS, IDI, or MVA pathway nucleic acids.

Exemplary Cell Culture Conditions

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Exemplary techniques maybe found in Manual of Methods for General Bacteriology Gerhardt et al.,eds), American Society for Microbiology, Washington, D.C. (1994) orBrock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., which areeach hereby incorporated by reference in their entireties, particularlywith respect to cell culture techniques. In some embodiments, the cellsare cultured in a culture medium under conditions permitting theexpression of one or more isoprene synthase, DXS, IDI, or MVA pathwaypolypeptides encoded by a nucleic acid inserted into the host cells.

Standard cell culture conditions can be used to culture the cells (see,for example, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to cell culture and fermentation conditions). Cells are grownand maintained at an appropriate temperature, gas mixture, and pH (suchas at about 20 to about 37° C., at about 6% to about 84% CO₂, and at apH between about 5 to about 9). In some embodiments, cells are grown at35° C. in an appropriate cell medium. In some embodiments, e.g.,cultures are cultured at approximately 28° C. in appropriate medium inshake cultures or fermentors until desired amount of isoprene productionis achieved. In some embodiments, the pH ranges for fermentation arebetween about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH8.0 or about 6.5 to about 7.0). Reactions may be performed underaerobic, anoxic, or anaerobic conditions based on the requirements ofthe host cells. Exemplary culture conditions for a given filamentousfungus are known in the art and may be found in the scientificliterature and/or from the source of the fungi such as the American TypeCulture Collection and Fungal Genetics Stock Center.

In various embodiments, the cells are grown using any known mode offermentation, such as batch, fed-batch, or continuous processes. In someembodiments, a batch method of fermentation is used. Classical batchfermentation is a closed system where the composition of the media isset at the beginning of the fermentation and is not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the cell medium is inoculated with the desired hostcells and fermentation is permitted to occur adding nothing to thesystem. Typically, however, “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems, the metabolite and biomass compositions of the system changeconstantly until the time the fermentation is stopped. Within batchcultures, cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. In some embodiments, cells in log phase are responsible forthe bulk of the isoprene production. In some embodiments, cells instationary phase produce isoprene.

In some embodiments, a variation on the standard batch system is used,such as the Fed-Batch system. Fed-Batch fermentation processes comprisea typical batch system with the exception that the carbon source isadded in increments as the fermentation progresses. Fed-Batch systemsare useful when catabolite repression is apt to inhibit the metabolismof the cells and where it is desirable to have limited amounts of carbonsource in the cell medium. Fed-batch fermentations may be performed withthe carbon source (e.g., glucose) in a limited or excess amount.Measurement of the actual carbon source concentration in Fed-Batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen, and thepartial pressure of waste gases such as CO₂. Batch and Fed-Batchfermentations are common and well known in the art and examples may befound in Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, continuous fermentation methods are used.Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or isoprene production. Forexample, one method maintains a limiting nutrient such as the carbonsource or nitrogen level at a fixed rate and allows all other parametersto moderate. In other systems, a number of factors affecting growth canbe altered continuously while the cell concentration (e.g., theconcentration measured by media turbidity) is kept constant. Continuoussystems strive to maintain steady state growth conditions. Thus, thecell loss due to media being drawn off is balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology and a variety of methods aredetailed by Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, cells are immobilized on a substrate as whole cellcatalysts and subjected to fermentation conditions for isopreneproduction.

In some embodiments, bottles of liquid culture are placed in shakers inorder to introduce oxygen to the liquid and maintain the uniformity ofthe culture. In some embodiments, an incubator is used to control thetemperature, humidity, shake speed, and/or other conditions in which aculture is grown. The simplest incubators are insulated boxes with anadjustable heater, typically going up to ˜65° C. More elaborateincubators can also include the ability to lower the temperature (viarefrigeration), or the ability to control humidity or CO₂ levels. Mostincubators include a timer; some can also be programmed to cycle throughdifferent temperatures, humidity levels, etc. Incubators can vary insize from tabletop to units the size of small rooms.

If desired, a portion or all of the cell medium can be changed toreplenish nutrients and/or avoid the build up of potentially harmfulmetabolic byproducts and dead cells. In the case of suspension cultures,cells can be separated from the media by centrifuging or filtering thesuspension culture and then resuspending the cells in fresh media. Inthe case of adherent cultures, the media can be removed directly byaspiration and replaced. In some embodiments, the cell medium allows atleast a portion of the cells to divide for at least or about 5, 10, 20,40, 50, 60, 65, or more cell divisions in a continuous culture (such asa continuous culture without dilution).

In some embodiments, a constitutive or leaky promoter (such as a Trcpromoter) is used and a compound (such as IPTG) is not added to induceexpression of the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid(s) operably linked to the promoter. In some embodiments, a compound(such as IPTG) is added to induce expression of the isoprene synthase,DXS, IDI, or MVA pathway nucleic acid(s) operably linked to thepromoter.

Exemplary Methods for Decoupling Isoprene Production from Cell Growth

Desirably, carbon from the feedstock is converted to isoprene ratherthan to the growth and maintenance of the cells. In some embodiments,the cells are grown to a low to medium OD₆₀₀, then production ofisoprene is started or increased. This strategy permits a large portionof the carbon to be converted to isoprene.

In some embodiments, cells reach an optical density such that they nolonger divide or divide extremely slowly, but continue to make isoprenefor several hours (such as about 2, 4, 6, 8, 10, 15, 20, 25, 30, or morehours). For example, FIGS. 60A-67C illustrate that cells may continue toproduce a substantial amount of mevalonic acid or isoprene after thecells reach an optical density such that they no longer divide or divideextremely slowly. In some cases, the optical density at 550 nm decreasesover time (such as a decrease in the optical density after the cells areno longer in an exponential growth phase due to cell lysis), and thecells continue to produce a substantial amount of mevalonic acid orisoprene. In some embodiments, the optical density at 550 nm of thecells increases by less than or about 50% (such as by less than or about40, 30, 20, 10, 5, or 0%) over a certain time period (such as greaterthan or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsproduce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750,2,000, 2,500, 3,000, 4,000, 5,000, or more nmole of isoprene/gram ofcells for the wet weight of the cells/hour (nmole/g_(wcm)/hr) duringthis time period. In some embodiments, the amount of isoprene is betweenabout 2 to about 5,000 nmole/g_(wcm)/hr, such as between about 2 toabout 100 nmole/g_(wcm)/hr, about 100 to about 500 nmole/g_(wcm)/hr,about 150 to about 500 nmole/g_(wcm)/hr, about 500 to about 1,000nmole/g_(wcm)/hr, about 1,000 to about 2,000 nmole/g_(wcm)/hr, or about2,000 to about 5,000 nmole/g_(wcm)/hr. In some embodiments, the amountof isoprene is between about 20 to about 5,000 nmole/g_(wcm)/hr, about100 to about 5,000 nmole/g_(wcm)/hr, about 200 to about 2,000nmole/g_(wcm)/hr, about 200 to about 1,000 nmole/g_(wcm)/hr, about 300to about 1,000 nmole/g_(wcm)/hr, or about 400 to about 1,000nmole/g_(wcm)/hr.

In some embodiments, the optical density at 550 nm of the cellsincreases by less than or about 50% (such as by less than or about 40,30, 20, 10, 5, or 0%) over a certain time period (such as greater thanor about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsproduce a cumulative titer (total amount) of isoprene at greater than orabout 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000,10,000, 50,000, 100,000, or more mg of isoprene/L of broth(mg/L_(broth), wherein the volume of broth includes the volume of thecells and the cell medium) during this time period. In some embodiments,the amount of isoprene is between about 2 to about 5,000 mg/L_(broth),such as between about 2 to about 100 mg/L_(broth), about 100 to about500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth), about 1,000 toabout 2,000 mg/L_(broth), or about 2,000 to about 5,000 mg/L_(broth). Insome embodiments, the amount of isoprene is between about 20 to about5,000 mg/L_(broth), about 100 to about 5,000 mg/L_(broth), about 200 toabout 2,000 mg/L_(broth), about 200 to about 1,000 mg/L_(broth), about300 to about 1,000 mg/L_(broth), or about 400 to about 1,000mg/L_(broth).

In some embodiments, the optical density at 550 nm of the cellsincreases by less than or about 50% (such as by less than or about 40,30, 20, 10, 5, or 0%) over a certain time period (such as greater thanor about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsconvert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05,0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2,1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of thecarbon in the cell culture medium into isoprene during this time period.In some embodiments, the percent conversion of carbon into isoprene isbetween such as about 0.002 to about 4.0%, about 0.002 to about 3.0%,about 0.002 to about 2.0%, about 0.002 to about 1.6%, about 0.002 toabout 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%,about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%,about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about1.0%, or about 1.0 to about 1.6%. In some embodiments, the percentconversion of carbon into isoprene is between about 0.002 to about 0.4%,0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to about 0.3%,about 0.01 to about 0.3%, or about 0.05 to about 0.3%.

In some embodiments, isoprene is only produced in stationary phase. Insome embodiments, isoprene is produced in both the growth phase andstationary phase. In various embodiments, the amount of isopreneproduced (such as the total amount of isoprene produced or the amount ofisoprene produced per liter of broth per hour per OD₆₀₀) duringstationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40,50, or more times the amount of isoprene produced during the growthphase for the same length of time. In various embodiments, greater thanor about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of thetotal amount of isoprene that is produced (such as the production ofisoprene during a fermentation for a certain amount of time, such as 20hours) is produced while the cells are in stationary phase. In variousembodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 95, 99% or more of the total amount of isoprene that is produced(such as the production of isoprene during a fermentation for a certainamount of time, such as 20 hours) is produced while the cells divideslowly or not at all such that the optical density at 550 nm of thecells increases by less than or about 50% (such as by less than or about40, 30, 20, 10, 5, or 0%). In some embodiments, isoprene is onlyproduced in the growth phase.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

Production of Isoprene within Safe Operating Ranges

The production of isoprene within safe operating levels according to itsflammability characteristics simplifies the design and construction ofcommercial facilities, vastly improves the ability to operate safely,and limits the potential for fires to occur. In particular, the optimalranges for the production of isoprene are within the safe zone, i.e.,the nonflammable range of isoprene concentrations. In one such aspect,the invention features a method for the production of isoprene withinthe nonflammable range of isoprene concentrations (outside theflammability envelope of isoprene).

Thus, computer modeling and experimental testing were used to determinethe flammability limits of isoprene (such as isoprene in the presence ofO₂, N₂, CO₂, or any combination of two or more of the foregoing gases)in order to ensure process safety. The flammability envelope ischaracterized by the lower flammability limit (LFL), the upperflammability limit (UFL), the limiting oxygen concentration (LOC), andthe limiting temperature. For a system to be flammable, a minimum amountof fuel (such as isoprene) must be in the presence of a minimum amountof oxidant, typically oxygen. The LFL is the minimum amount of isoprenethat must be present to sustain burning, while the UFL is the maximumamount of isoprene that can be present. Above this limit, the mixture isfuel rich and the fraction of oxygen is too low to have a flammablemixture. The LOC indicates the minimum fraction of oxygen that must alsobe present to have a flammable mixture. The limiting temperature isbased on the flash point of isoprene and is that lowest temperature atwhich combustion of isoprene can propagate. These limits are specific tothe concentration of isoprene, type and concentration of oxidant, inertspresent in the system, temperature, and pressure of the system.Compositions that fall within the limits of the flammability envelopepropagate combustion and require additional safety precautions in boththe design and operation of process equipment.

The following conditions were tested using computer simulation andmathematical analysis and experimental testing. If desired, otherconditions (such as other temperature, pressure, and permanent gascompositions) may be tested using the methods described herein todetermine the LFL, UFL, and LOC concentrations.

(1) Computer Simulation and Mathematical Analysis

Test Suite 1:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

Test Suite 2:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

Saturated with H₂O

Test Suite 3:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

CO₂: 5 wt %-30 wt %

(2) Experimental Testing for Final Determination of Flammability Limits

Test Suite 1:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

Test Suite 2:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

Saturated with H₂O

Simulation software was used to give an estimate of the flammabilitycharacteristics of the system for several different testing conditions.CO₂ showed no significant affect on the system's flammability limits.Test suites 1 and 2 were confirmed by experimental testing. The modelingresults were in-line with the experimental test results. Only slightvariations were found with the addition of water.

The LOC was determined to be 9.5 vol % for an isoprene, O₂, N₂, and CO₂mixture at 40° C. and 1 atmosphere. The addition of up to 30% CO₂ didnot significantly affect the flammability characteristics of anisoprene, O₂, and N₂ mixture. Only slight variations in flammabilitycharacteristics were shown between a dry and water saturated isoprene,O₂, and N₂ system. The limiting temperature is about −54° C.Temperatures below about −54° C. are too low to propagate combustion ofisoprene.

In some embodiments, the LFL of isoprene ranges from about 1.5 vol. % toabout 2.0 vol %, and the UFL of isoprene ranges from about 2.0 vol. % toabout 12.0 vol. %, depending on the amount of oxygen in the system. Insome embodiments, the LOC is about 9.5 vol % oxygen. In someembodiments, the LFL of isoprene is between about 1.5 vol. % to about2.0 vol %, the UFL of isoprene is between about 2.0 vol. % to about 12.0vol. %, and the LOC is about 9.5 vol % oxygen when the temperature isbetween about 25° C. to about 55° C. (such as about 40° C.) and thepressure is between about 1 atmosphere and 3 atmospheres.

In some embodiments, isoprene is produced in the presence of less thanabout 9.5 vol % oxygen (that is, below the LOC required to have aflammable mixture of isoprene). In some embodiments in which isoprene isproduced in the presence of greater than or about 9.5 vol % oxygen, theisoprene concentration is below the LFL (such as below about 1.5 vol.%). For example, the amount of isoprene can be kept below the LFL bydiluting the isoprene composition with an inert gas (e.g., bycontinuously or periodically adding an inert gas such as nitrogen tokeep the isoprene composition below the LFL). In some embodiments inwhich isoprene is produced in the presence of greater than or about 9.5vol % oxygen, the isoprene concentration is above the UFL (such as aboveabout 12 vol. %). For example, the amount of isoprene can be kept abovethe UFL by using a system (such as any of the cell culture systemsdescribed herein) that produces isoprene at a concentration above theUFL. If desired, a relatively low level of oxygen can be used so thatthe UFL is also relatively low. In this case, a lower isopreneconcentration is needed to remain above the UFL.

In some embodiments in which isoprene is produced in the presence ofgreater than or about 9.5 vol % oxygen, the isoprene concentration iswithin the flammability envelope (such as between the LFL and the UFL).In some embodiments when the isoprene concentration may fall within theflammability envelope, one or more steps are performed to reduce theprobability of a fire or explosion. For example, one or more sources ofignition (such as any materials that may generate a spark) can beavoided. In some embodiments, one or more steps are performed to reducethe amount of time that the concentration of isoprene remains within theflammability envelope. In some embodiments, a sensor is used to detectwhen the concentration of isoprene is close to or within theflammability envelope. If desired, the concentration of isoprene can bemeasured at one or more time points during the culturing of cells, andthe cell culture conditions and/or the amount of inert gas can beadjusted using standard methods if the concentration of isoprene isclose to or within the flammability envelope. In particular embodiments,the cell culture conditions (such as fermentation conditions) areadjusted to either decrease the concentration of isoprene below the LFLor increase the concentration of isoprene above the UFL. In someembodiments, the amount of isoprene is kept below the LFL by dilutingthe isoprene composition with an inert gas (such as by continuously orperiodically adding an inert gas to keep the isoprene composition belowthe LFL).

In some embodiments, the amount of flammable volatiles other thanisoprene (such as one or more sugars) is at least about 2, 5, 10, 50,75, or 100-fold less than the amount of isoprene produced. In someembodiments, the portion of the gas phase other than isoprene gascomprises between about 0% to about 100% (volume) oxygen, such asbetween about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 90% toabout 90%, or about 90% to about 100% (volume) oxygen. In someembodiments, the portion of the gas phase other than isoprene gascomprises between about 0% to about 99% (volume) nitrogen, such asbetween about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 90% toabout 90%, or about 90% to about 99% (volume) nitrogen.

In some embodiments, the portion of the gas phase other than isoprenegas comprises between about 1% to about 50% (volume) CO₂, such asbetween about 1% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, or about 40% to about 50% (volume)CO₂.

In some embodiments, an isoprene composition also contains ethanol. Forexample, ethanol may be used for extractive distillation of isoprene,resulting in compositions (such as intermediate product streams) thatinclude both ethanol and isoprene. Desirably, the amount of ethanol isoutside the flammability envelope for ethanol. The LOC of ethanol isabout 8.7 vol %, and the LFL for ethanol is about 3.3 vol % at standardconditions, such as about 1 atmosphere and about 60° F. (NFPA 69Standard on Explosion Prevention Systems, 2008 edition, which is herebyincorporated by reference in its entirety, particularly with respect toLOC, LFL, and UFL values). In some embodiments, compositions thatinclude isoprene and ethanol are produced in the presence of less thanthe LOC required to have a flammable mixture of ethanol (such as lessthan about 8.7% vol %). In some embodiments in which compositions thatinclude isoprene and ethanol are produced in the presence of greaterthan or about the LOC required to have a flammable mixture of ethanol,the ethanol concentration is below the LFL (such as less than about 3.3vol. %).

In various embodiments, the amount of oxidant (such as oxygen) is belowthe LOC of any fuel in the system (such as isoprene or ethanol). Invarious embodiments, the amount of oxidant (such as oxygen) is less thanabout 60, 40, 30, 20, 10, or 5% of the LOC of isoprene or ethanol. Invarious embodiments, the amount of oxidant (such as oxygen) is less thanthe LOC of isoprene or ethanol by at least 2, 4, 5, or more absolutepercentage points (vol %). In particular embodiments, the amount ofoxygen is at least 2 absolute percentage points (vol %) less than theLOC of isoprene or ethanol (such as an oxygen concentration of less than7.5 vol % when the LOC of isoprene is 9.5 vol %). In variousembodiments, the amount of fuel (such as isoprene or ethanol) is lessthan or about 25, 20, 15, 10, or 5% of the LFL for that fuel.

Exemplary Production of Bioisoprene

In some embodiments, the cells are cultured in a culture medium underconditions permitting the production of isoprene by the cells. By “peakabsolute productivity” is meant the maximum absolute amount of isoprenein the off-gas during the culturing of cells for a particular period oftime (e.g., the culturing of cells during a particular fermentationrun). By “peak absolute productivity time point” is meant the time pointduring a fermentation run when the absolute amount of isoprene in theoff-gas is at a maximum during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the isoprene amount is measuredat the peak absolute productivity time point. In some embodiments, thepeak absolute productivity for the cells is about any of the isopreneamounts disclosed herein.

By “peak specific productivity” is meant the maximum amount of isopreneproduced per cell during the culturing of cells for a particular periodof time (e.g., the culturing of cells during a particular fermentationrun). By “peak specific productivity time point” is meant the time pointduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run) when the amountof isoprene produced per cell is at a maximum. The peak specificproductivity is determined by dividing the total productivity by theamount of cells, as determined by optical density at 600 nm (OD₆₀₀). Insome embodiments, the isoprene amount is measured at the peak specificproductivity time point. In some embodiments, the peak specificproductivity for the cells is about any of the isoprene amounts per celldisclosed herein.

By “peak volumetric productivity” is meant the maximum amount ofisoprene produced per volume of broth (including the volume of the cellsand the cell medium) during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). By “peak specific volumetric productivity time point”is meant the time point during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run) when the amount of isoprene produced per volume ofbroth is at a maximum. The peak specific volumetric productivity isdetermined by dividing the total productivity by the volume of broth andamount of time. In some embodiments, the isoprene amount is measured atthe peak specific volumetric productivity time point. In someembodiments, the peak specific volumetric productivity for the cells isabout any of the isoprene amounts per volume per time disclosed herein.

By “peak concentration” is meant the maximum amount of isoprene producedduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run). By “peakconcentration time point” is meant the time point during the culturingof cells for a particular period of time (e.g., the culturing of cellsduring a particular fermentation run) when the amount of isopreneproduced per cell is at a maximum. In some embodiments, the isopreneamount is measured at the peak concentration time point. In someembodiments, the peak concentration for the cells is about any of theisoprene amounts disclosed herein.

By “average volumetric productivity” is meant the average amount ofisoprene produced per volume of broth (including the volume of the cellsand the cell medium) during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). The average volumetric productivity is determined bydividing the total productivity by the volume of broth and amount oftime. In some embodiments, the average specific volumetric productivityfor the cells is about any of the isoprene amounts per volume per timedisclosed herein.

By “cumulative total productivity” is meant the cumulative, total amountof isoprene produced during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the cumulative, total amount ofisoprene is measured. In some embodiments, the cumulative totalproductivity for the cells is about any of the isoprene amountsdisclosed herein.

As used herein, “relative detector response” refers to the ratio betweenthe detector response (such as the GC/MS area) for one compound (such asisoprene) to the detector response (such as the GC/MS area) of one ormore compounds (such as all C5 hydrocarbons). The detector response maybe measured as described herein, such as the GC/MS analysis performedwith an Agilent 6890 GC/MS system fitted with an Agilent HP-5MS GC/MScolumn (30 m×250 μm; 0.25 μm film thickness). If desired, the relativedetector response can be converted to a weight percentage using theresponse factors for each of the compounds. This response factor is ameasure of how much signal is generated for a given amount of aparticular compound (that is, how sensitive the detector is to aparticular compound). This response factor can be used as a correctionfactor to convert the relative detector response to a weight percentagewhen the detector has different sensitivities to the compounds beingcompared. Alternatively, the weight percentage can be approximated byassuming that the response factors are the same for the compounds beingcompared. Thus, the weight percentage can be assumed to be approximatelythe same as the relative detector response.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000,125,000, 150,000, 188,000, or more nmole of isoprene/gram of cells forthe wet weight of the cells/hour (nmole/g_(wcm)/hr). In someembodiments, the amount of isoprene is between about 2 to about 200,000nmole/g_(wcm)/hr, such as between about 2 to about 100 nmole/g_(wcm)/hr,about 100 to about 500 nmole/g_(wcm)/hr, about 150 to about 500nmole/g_(wcm)/hr, about 500 to about 1,000 nmole/g_(wcm)/hr, about 1,000to about 2,000 nmole/g_(wcm)/hr, about 2,000 to about 5,000nmole/g_(wcm)/hr, about 5,000 to about 10,000 nmole/g_(wcm)/hr, about10,000 to about 50,000 nmole/g_(wcm)/hr, about 50,000 to about 100,000nmole/g_(wcm)/hr, about 100,000 to about 150,000 nmole/g_(wcm)/hr, orabout 150,000 to about 200,000 nmole/g_(wcm)/hr. In some embodiments,the amount of isoprene is between about 20 to about 200,000nmole/g_(wcm)/hr, about 100 to about 5,000 nmole/g_(wcm)/hr, about 200to about 2,000 nmole/g_(wcm)/hr, about 200 to about 1,000nmole/g_(wcm)/hr, about 300 to about 1,000 nmole/g_(wcm)/hr, about 400to about 1,000 nmole/g_(wcm)/hr, about 1,000 to about 5,000nmole/g_(wcm)/hr, about 2,000 to about 20,000 nmole/g_(wcm)/hr, about5,000 to about 50,000 nmole/g_(wcm)/hr, about 10,000 to about 100,000nmole/g_(wcm)/hr, about 20,000 to about 150,000 nmole/g_(wcm)/hr, orabout 20,000 to about 200,000 nmole/g_(wcm)/hr.

The amount of isoprene in units of nmole/g_(wcm)/hr can be measured asdisclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated byreference in its entirety, particularly with respect to the measurementof isoprene production. For example, two mL of headspace (e.g.,headspace from a culture such as 2 mL of culture cultured in sealedvials at 32° C. with shaking at 200 rpm for approximately 3 hours) areanalyzed for isoprene using a standard gas chromatography system, suchas a system operated isothermally (85° C.) with an n-octane/porasil Ccolumn (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2mercuric oxide reduction gas detector (Trace Analytical, Menlo Park,Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A:2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to the measurement of isoprene production).The gas chromatography area units are converted to nmol isoprene via astandard isoprene concentration calibration curve. In some embodiments,the value for the grams of cells for the wet weight of the cells iscalculated by obtaining the A₆₀₀ value for a sample of the cell culture,and then converting the A₆₀₀ value to grams of cells based on acalibration curve of wet weights for cell cultures with a known A₆₀₀value. In some embodiments, the grams of the cells is estimated byassuming that one liter of broth (including cell medium and cells) withan A₆₀₀ value of 1 has a wet cell weight of 1 gram. The value is alsodivided by the number of hours the culture has been incubating for, suchas three hours.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wetweight of the cells/hr (ng/g_(wcm)/h). In some embodiments, the amountof isoprene is between about 2 to about 5,000 ng/g_(wcm)/h, such asbetween about 2 to about 100 ng/g_(wcm)/h, about 100 to about 500ng/g_(wcm)/h, about 500 to about 1,000 ng/g_(wcm)/h, about 1,000 toabout 2,000 ng/g_(wcm)/h, or about 2,000 to about 5,000 ng/g_(wcm)/h. Insome embodiments, the amount of isoprene is between about 20 to about5,000 ng/g_(wcm)/h, about 100 to about 5,000 ng/g_(wcm)/h, about 200 toabout 2,000 ng/g_(wcm)/h, about 200 to about 1,000 ng/g_(wcm)/h, about300 to about 1,000 ng/g_(wcm)/h, or about 400 to about 1,000ng/g_(wcm)/h. The amount of isoprene in ng/g_(wcm)/h can be calculatedby multiplying the value for isoprene production in the units ofnmole/g_(wcm)/hr discussed above by 68.1 (as described in Equation 5below).

In some embodiments, the cells in culture produce a cumulative titer(total amount) of isoprene at greater than or about 1, 10, 25, 50, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, ormore mg of isoprene/L of broth (mg/L_(broth), wherein the volume ofbroth includes the volume of the cells and the cell medium). In someembodiments, the amount of isoprene is between about 2 to about 5,000mg/L_(broth), such as between about 2 to about 100 mg/L_(broth), about100 to about 500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth),about 1,000 to about 2,000 mg/L_(broth), or about 2,000 to about 5,000mg/L_(broth). In some embodiments, the amount of isoprene is betweenabout 20 to about 5,000 mg/L_(broth), about 100 to about 5,000mg/L_(broth), about 200 to about 2,000 mg/L_(broth), about 200 to about1,000 mg/L_(broth), about 300 to about 1,000 mg/L_(broth), or about 400to about 1,000 mg/L_(broth).

The specific productivity of isoprene in mg of isoprene/L of headspacefrom shake flask or similar cultures can be measured by taking a 1 mlsample from the cell culture at an OD₆₀₀ value of approximately 1.0,putting it in a 20 mL vial, incubating for 30 minutes, and thenmeasuring the amount of isoprene in the headspace (as described, forexample, in Example I, part II). If the OD₆₀₀ value is not 1.0, then themeasurement can be normalized to an OD₆₀₀ value of 1.0 by dividing bythe OD₆₀₀ value. The value of mg isoprene/L headspace can be convertedto mg/L_(broth)/hr/OD₆₀₀ of culture broth by multiplying by a factor of38. The value in units of mg/L_(broth)/hr/OD₆₀₀ can be multiplied by thenumber of hours and the OD₆₀₀ value to obtain the cumulative titer inunits of mg of isoprene/L of broth.

In some embodiments, the cells in culture have an average volumetricproductivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr(mg/L_(broth)/hr, wherein the volume of broth includes the volume of thecells and the cell medium). In some embodiments, the average volumetricproductivity of isoprene is between about 0.1 to about 3,500mg/L_(broth)/hr, such as between about 0.1 to about 100 mg/L_(broth)/hr,about 100 to about 500 mg/L_(broth)/hr, about 500 to about 1,000mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr, about 1,500to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, or about3,000 to about 3,500 mg/L_(broth)/hr. In some embodiments, the averagevolumetric productivity of isoprene is between about 10 to about 3,500mg/L_(broth)/hr, about 100 to about 3,500 mg/L_(broth)/hr, about 200 toabout 1,000 mg/L_(broth)/hr, about 200 to about 1,500 mg/L_(broth)/hr,about 1,000 to about 3,000 mg/L_(broth)/hr, or about 1,500 to about3,000 mg/L_(broth)/hr.

In some embodiments, the cells in culture have a peak volumetricproductivity of isoprene at greater than or about 0.5, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000,5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000, 7,250, 7,500,7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500, 9,750, 10,000,12,500, 15,000, or more mg of isoprene/L of broth/hr (mg/L_(broth)/hr,wherein the volume of broth includes the volume of the cells and thecell medium). In some embodiments, the peak volumetric productivity ofisoprene is between about 0.5 to about 15,000 mg/L_(broth)/hr, such asbetween about 0.5 to about 10 mg/L_(broth)/hr, about 1.0 to about 100mg/L_(broth)/hr, about 100 to about 500 mg/L_(broth)/hr, about 500 toabout 1,000 mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr,about 1,500 to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, about 3,000to about 3,500 mg/L_(broth)/hr, about 3,500 to about 5,000mg/L_(broth)/hr, about 5,000 to about 7,500 mg/L_(broth)/hr, about 7,500to about 10,000 mg/L_(broth)/hr, about 10,000 to about 12,500mg/L_(broth)/h, or about 12,500 to about 15,000 mg/L_(broth)/hr. In someembodiments, the peak volumetric productivity of isoprene is betweenabout 10 to about 15,000 mg/L_(broth)/hr, about 100 to about 2,500mg/L_(broth)/hr, about 1,000 to about 5,000 mg/L_(broth)/hr, about 2,500to about 7,500 mg/L_(broth)/hr, about 5,000 to about 10,000mg/L_(broth)/hr, about 7,500 to about 12,500 mg/L_(broth)/hr, or about10,000 to about 15,000 mg/L_(broth)/hr.

The instantaneous isoprene production rate in mg/L_(broth)/hr in afermentor can be measured by taking a sample of the fermentor off-gas,analyzing it for the amount of isoprene (in units such as mg of isopreneper L_(gas)) as described, for example, in Example I, part II andmultiplying this value by the rate at which off-gas is passed thougheach liter of broth (e.g., at 1 vvm (volume of air/volume ofbroth/minute) this is 60 L_(gas) per hour). Thus, an off-gas level of 1mg/L_(gas) corresponds to an instantaneous production rate of 60mg/L_(broth)/hr at air flow of 1 vvm. If desired, the value in units ofmg/L_(broth)/hr can be divided by the OD₆₀₀ value to obtain the specificrate in units of mg/L_(broth)/hr/OD. The average value of mgisoprene/L_(gas) can be converted to the total product productivity(grams of isoprene per liter of fermentation broth, mg/L_(broth)) bymultiplying this average off-gas isoprene concentration by the totalamount of off-gas sparged per liter of fermentation broth during thefermentation. Thus, an average off-gas isoprene concentration of 0.5mg/L_(broth)/hr over 10 hours at 1 vvm corresponds to a total productconcentration of 300 mg isoprene/L_(broth).

In some embodiments, the cells in culture convert greater than or about0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 2.0, 2.2, 2.4, 2.6,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0,16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6, 23.8,24.0, 25.0, 30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5, 50.0,55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, or 90.0 molar % of the carbonin the cell culture medium into isoprene. In some embodiments, thepercent conversion of carbon into isoprene is between about 0.002 toabout 90.0 molar %, such as about 0.002 to about 0.005%, about 0.005 toabout 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about0.5 to about 0.8%, about 0.8 to about 1.0%, about 1.0 to about 1.6%,about 1.6 to about 3.0%, about 3.0 to about 5.0%, about 5.0 to about8.0%, about 8.0 to about 10.0%, about 10.0 to about 15.0%, about 15.0 toabout 20.0%, about 20.0 to about 25.0%, about 25.0% to 30.0%, about30.0% to 35.0%, about 35.0% to 40.0%, about 45.0% to 50.0%, about 50.0%to 55.0%, about 55.0% to 60.0%, about 60.0% to 65.0%, about 65.0% to70.0%, about 75.0% to 80.0%, about 80.0% to 85.0%, or about 85.0% to90.0%. In some embodiments, the percent conversion of carbon intoisoprene is between about 0.002 to about 0.4 molar %, 0.002 to about0.16 molar %, 0.04 to about 0.16 molar %, about 0.005 to about 0.3 molar%, about 0.01 to about 0.3 molar %, about 0.05 to about 0.3 molar %,about 0.1 to 0.3 molar %, about 0.3 to about 1.0 molar %, about 1.0 toabout 5.0 molar %, about 2 to about 5.0 molar %, about 5.0 to about 10.0molar %, about 7 to about 10.0 molar %, about 10.0 to about 20.0 molar%, about 12 to about 20.0 molar %, about 16 to about 20.0 molar %, about18 to about 20.0 molar %, about 18 to 23.2 molar %, about 18 to 23.6molar %, about 18 to about 23.8 molar %, about 18 to about 24.0 molar %,about 18 to about 25.0 molar %, about 20 to about 30.0 molar %, about 30to about 40.0 molar %, about 30 to about 50.0 molar %, about 30 to about60.0 molar %, about 30 to about 70.0 molar %, about 30 to about 80.0molar %, or about 30 to about 90.0 molar %.

The percent conversion of carbon into isoprene (also referred to as “%carbon yield”) can be measured by dividing the moles carbon in theisoprene produced by the moles carbon in the carbon source (such as themoles of carbon in batched and fed glucose and yeast extract). Thisnumber is multiplied by 100% to give a percentage value (as indicated inEquation 1).

$\begin{matrix}{{{Carbon}\mspace{14mu}{Yield}} = \frac{{moles}\mspace{14mu}{carbon}\mspace{14mu}{in}\mspace{14mu}{isoprene}\mspace{14mu}{produced}}{\left( {{moles}\mspace{14mu}{carbon}\mspace{14mu}{in}\mspace{14mu}{carbon}\mspace{14mu}{source}} \right) \times 100}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

For this calculation, yeast extract can be assumed to contain 50% w/wcarbon. As an example, for the 500 liter described in Example 7, partVIII, the percent conversion of carbon into isoprene can be calculatedas shown in Equation 2.

$\begin{matrix}\begin{matrix}{{\%\mspace{14mu}{Carbon}\mspace{14mu}{Yield}} = \frac{39.1\mspace{14mu} g\mspace{14mu}{isoprene} \times {1/68.1}\mspace{14mu}{{mol}/g} \times 5{C/{mol}}}{\left\lbrack {\left( {181.221\mspace{14mu} g\mspace{14mu}{glucose} \times {1/180}\mspace{14mu}{{mol}/g} \times 6\mspace{14mu}{C/{mol}}} \right) + \left( {17,{780\mspace{14mu} g\mspace{14mu}{yeast}\mspace{14mu}{extract} \times 0.5 \times {1/12}\mspace{14mu}{{mol}/g}}} \right)} \right\rbrack \times 100}} \\{= {0.042\mspace{14mu}\%}}\end{matrix} & {{Equation}\mspace{14mu} 2}\end{matrix}$

For the two 500 liter fermentations described herein (Example 7, partsVII and VIII), the percent conversion of carbon into isoprene wasbetween 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14liter systems as described herein.

One skilled in the art can readily convert the rates of isopreneproduction or amount of isoprene produced into any other units.Exemplary equations are listed below for interconverting between units.

Units for Rate of Isoprene Production (Total and Specific)1 g isoprene/L_(broth)/hr=14.7 mmol isoprene/L_(broth)/hr(totalvolumetric rate)  Equation 31 nmol isoprene/g_(wcm)/hr=1 nmol isoprene/L_(broth)/hr/OD₆₀₀  Equation4(This conversion assumes that one liter of broth with an OD₆₀₀ value of1 has a wet cell weight of 1 gram.)1 nmol isoprene/g_(wcm)/hr=68.1 ng isoprene/g_(wcm)/hr(given themolecular weight of isoprene)  Equation 51 nmol isoprene/L_(gas)O₂/hr=90 nmol isoprene/L_(broth)/hr(at an O₂ flowrate of 90 L/hr per L of culture broth)  Equation 61 μg isoprene/L_(gas) isoprene in off-gas=60 μg isoprene/L_(broth)/hr ata flow rate of 60 L_(gas) per L_(broth)(1 vvm)  Equation 7Units for Titer (Total and Specific)1 nmol isoprene/mg cell protein=150 nmolisoprene/L_(broth)/OD₆₀₀  Equation 8(This conversion assumes that one liter of broth with an OD₆₀₀ value of1 has a total cell protein of approximately 150 mg) (specificproductivity)1 g isoprene/L_(broth)=14.7 mmol isoprene/L_(broth)(totaltiter)  Equation 9

If desired, Equation 10 can be used to convert any of the units thatinclude the wet weight of the cells into the corresponding units thatinclude the dry weight of the cells.Dry weight of cells=(wet weight of cells)/3.3  Equation 10

If desired, Equation 11 can be used to convert between units of ppm andμg/L. In particular, “ppm” means parts per million defined in terms ofμg/g (w/w). Concentrations of gases can also be expressed on avolumeteic basis using “ppmv” (parts per million by volume), defined interms of μL/L (vol/vol). Conversion of μg/L to ppm (e.g., μg of analyteper g of gas) can be performed by determining the mass per L of off-gas(i.e., the density of the gas). For example, a liter of air at standardtemperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K) has adensity of approximately 1.29 g/L. Thus, a concentration of 1 ppm (μg/g)equals 1.29 μg/L at STP (equation 11). The conversion of ppm (μg/g) toμg/L is a function of both pressure, temperature, and overallcomposition of the off-gas.1 ppm(μg/g)equals 1.29 μg/L at standard temperature andpressure(STP;101.3 kPa(1 bar) and 273.15K).  Equation 11

Conversion of μg/L to ppmv (e.g., μL of analyte per L of gas) can beperformed using the Universal Gas Law (equation 12). For example, anoff-gas concentration of 1000 μg/L_(gas) corresponds to 14.7μmol/L_(gas). The universal gas constant is 0.082057 L·atm K⁻¹ mol⁻¹, sousing equation 12, the volume occupied by 14.7 μmol of HG at STP isequal to 0.329 mL. Therefore, the concentration of 1000 μg/L HG is equalto 329 ppmv or 0.0329% (v/v) at STP.PV=nRT,  Equation 12where “P” is pressure, “V” is volume, “n” is moles of gas, “R” is theUniversal gas constant, and “T” is temperature in Kelvin.

The amount of impurities in isoprene compositions are typically measuredherein on a weight per volume (w/v) basis in units such as μg/L. Ifdesired, measurements in units of μg/L can be converted to units ofmg/m³ using equation 13.1 μg/L=1 mg/m³  Equation 13

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptideproduces an amount of isoprene that is at least or about 2-fold, 3-fold,5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold,400-fold, or greater than the amount of isoprene produced from acorresponding cell grown under essentially the same conditions withoutthe heterologous nucleic acid encoding the isoprene synthasepolypeptide.

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptide andone or more heterologous nucleic acids encoding a DXS, IDI, and/or MVApathway polypeptide produces an amount of isoprene that is at least orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of isopreneproduced from a corresponding cell grown under essentially the sameconditions without the heterologous nucleic acids.

In some embodiments, the isoprene composition comprises greater than orabout 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.In some embodiments, the composition has a relative detector response ofgreater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96,99.97, 99.98, 99.99, or 100% for isoprene compared to the detectorresponse for all C5 hydrocarbons in the composition. In someembodiments, the isoprene composition comprises between about 99.90 toabout 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96,about 99.96 to about 99.98, about 99.98 to 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the isoprene composition comprises less than orabout 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005,0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the composition. In some embodiments, the compositionhas a relative detector response of less than or about 0.12, 0.10, 0.08,0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or0.00001% for C5 hydrocarbons other than isoprene compared to thedetector response for all C5 hydrocarbons in the composition. In someembodiments, the composition has a relative detector response of lessthan or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001,0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-ynecompared to the detector response for all C5 hydrocarbons in thecomposition. In some embodiments, the isoprene composition comprisesbetween about 0.02 to about 0.04%, about 0.04 to about 0.06%, about 0.06to 0.08%, about 0.08 to 0.10%, or about 0.10 to about 0.12% C5hydrocarbons other than isoprene (such 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) byweight compared to the total weight of all C5 hydrocarbons in thecomposition.

In some embodiments, the isoprene composition comprises less than orabout 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of acompound that inhibits the polymerization of isoprene for any compoundin the composition that inhibits the polymerization of isoprene. In someembodiments, the isoprene composition comprises between about 0.005 toabout 50, such as about 0.01 to about 10, about 0.01 to about 5, about0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005μg/L of a compound that inhibits the polymerization of isoprene for anycompound in the composition that inhibits the polymerization ofisoprene. In some embodiments, the isoprene composition comprises lessthan or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005μg/L of a hydrocarbon other than isoprene (such 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne). Insome embodiments, the isoprene composition comprises between about 0.005to about 50, such as about 0.01 to about 10, about 0.01 to about 5,about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about0.005 μg/L of a hydrocarbon other than isoprene. In some embodiments,the isoprene composition comprises less than or about 50, 40, 30, 20,10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a protein or fatty acid(such as a protein or fatty acid that is naturally associated withnatural rubber).

In some embodiments, the isoprene composition comprises less than orabout 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alphaacetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In someembodiments, the isoprene composition comprises less than or about 5, 1,0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In someembodiments, the isoprene composition comprises less than or about 30,20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes(such as 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne). In some embodiments,the isoprene composition comprises less than or about 2000, 1000, 500,200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppmof isoprene dimers, such as cyclic isoprene dimmers (e.g., cyclic C10compounds derived from the dimerization of two isoprene units).

In some embodiments, the isoprene composition includes ethanol, acetone,a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or3-methyl-2-buten-1-ol), or any two or more of the foregoing. Inparticular embodiments, the isoprene composition comprises greater thanor about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100,or 120 μg/L of ethanol, acetone, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more ofthe foregoing. In some embodiments, the isoprene composition comprisesbetween about 0.005 to about 120, such as about 0.01 to about 80, about0.01 to about 60, about 0.01 to about 40, about 0.01 to about 30, about0.01 to about 20, about 0.01 to about 10, about 0.1 to about 80, about0.1 to about 60, about 0.1 to about 40, about 5 to about 80, about 5 toabout 60, or about 5 to about 40 μg/L of ethanol, acetone, a C5 prenylalcohol, or any two or more of the foregoing.

In some embodiments, the isoprene composition includes one or more ofthe following components: 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl,2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or alinear isoprene polymer (such as a linear isoprene dimer or a linearisoprene trimer derived from the polymerization of multiple isopreneunits). In various embodiments, the amount of one of these componentsrelative to amount of isoprene in units of percentage by weight (i.e.,weight of the component divided by the weight of isoprene times 100) isgreater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments, therelative detector response for the second compound compared to thedetector response for isoprene is greater than or about 0.01, 0.02,0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%.In various embodiments, the amount of one of these components relativeto amount of isoprene in units of percentage by weight (i.e., weight ofthe component divided by the weight of isoprene times 100) is betweenabout 0.01 to about 105% (w/w), such as about 0.01 to about 90, about0.01 to about 80, about 0.01 to about 50, about 0.01 to about 20, about0.01 to about 10, about 0.02 to about 50, about 0.05 to about 50, about0.1 to about 50, or 0.1 to about 20% (w/w).

In some embodiments, the isoprene composition includes one or more ofthe following: an alcohol, an aldehyde, or a ketone (such as any of thealcohols, aldehydes, or ketones described herein). In some embodiments,the isoprene composition includes (i) an alcohol and an aldehyde, (ii)an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) analcohol, an aldehyde, and a ketone.

In some embodiments, the isoprene composition contains one or more ofthe following: methanol, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the isoprenecomposition contains 1 ppm or more of one or more of the following:methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the concentration ofmore of one or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole, is between about 1 to about10,000 ppm in an isoprene composition (such as off-gas before it ispurified). In some embodiments, the isoprene composition (such asoff-gas after it has undergone one or more purification steps) includesone or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole, at a concentration betweenabout 1 to about 100 ppm, such as about 1 to about 10 ppm, about 10 toabout 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm, about40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70 ppm,about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 to about100 ppm. Volatile organic compounds from cell cultures (such as volatileorganic compounds in the headspace of cell cultures) can be analyzedusing standard methods such as those described herein or other standardmethods such as proton transfer reaction-mass spectrometry (see, forexample, Bunge et al., Applied and Environmental Microbiology,74(7):2179-2186, 2008 which is hereby incorporated by reference in itsentirety, particular with respect to the analysis of volatile organiccompounds).

In some embodiments, the composition comprises greater than about 2 mgof isoprene, such as greater than or about 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg ofisoprene. In some embodiments, the composition comprises greater than orabout 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. Insome embodiments, the amount of isoprene in the composition is betweenabout 2 to about 5,000 mg, such as between about 2 to about 100 mg,about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 toabout 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments,the amount of isoprene in the composition is between about 20 to about5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg,about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400to about 1,000 mg. In some embodiments, greater than or about 20, 25,30, 40, 50, 60, 70, 80, 90, or 95% by weight of the volatile organicfraction of the composition is isoprene.

In some embodiments, the composition includes ethanol. In someembodiments, the composition includes between about 75 to about 90% byweight of ethanol, such as between about 75 to about 80%, about 80 toabout 85%, or about 85 to about 90% by weight of ethanol. In someembodiments in which the composition includes ethanol, the compositionalso includes between about 4 to about 15% by weight of isoprene, suchas between about 4 to about 8%, about 8 to about 12%, or about 12 toabout 15% by weight of isoprene.

In some embodiments encompassed by the invention, a cell comprising oneor more heterologous nucleic acids encoding an isoprene synthasepolypeptide, DXS polypeptide, IDI polypeptide, and/or MVA pathwaypolypeptide produces an amount of an isoprenoid compound (such as acompound with 10 or more carbon atoms that is formed from the reactionof one or more IPP molecules with one or more DMAPP molecules) that isgreater than or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold,100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount ofthe isoprenoid compound produced from a corresponding cell grown underessentially the same conditions without the one or more heterologousnucleic acids. In some embodiments encompassed by the invention, a cellcomprising one or more heterologous nucleic acids encoding an isoprenesynthase polypeptide, DXS polypeptide, IDI polypeptide, and/or MVApathway polypeptide produces an amount of a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol) that is greater than orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of the C5prenyl alcohol produced from a corresponding cell grown underessentially the same conditions without the one or more heterologousnucleic acids.

Exemplary Isoprene Purification Methods

In some embodiments, any of the methods described herein further includerecovering the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be recovered usingstandard techniques, such as gas stripping, fractionation,adsorption/desorption, pervaporation, thermal or vacuum desorption ofisoprene from a solid phase, or extraction of isoprene immobilized orabsorbed to a solid phase with a solvent (see, for example, U.S. Pat.No. 4,703,007 and U.S. Pat. No. 4,570,029, which are each herebyincorporated by reference in their entireties, particularly with respectto isoprene recovery and purification methods). In some embodiments, therecovery of isoprene involves the isolation of isoprene in a liquid form(such as a neat solution of isoprene or a solution of isoprene in asolvent). Gas stripping involves the removal of isoprene vapor from thefermentation off-gas stream in a continuous manner. Such removal can beachieved in several different ways including, but not limited to,adsorption to a solid phase, partition into a liquid phase, or directcondensation. In some embodiments, membrane enrichment of a diluteisoprene vapor stream above the dew point of the vapor resulting in thecondensation of liquid isoprene.

The recovery of isoprene may involve one step or multiple steps. In someembodiments, the removal of isoprene vapor from the fermentation off-gasand the conversion of isoprene to a liquid phase are performedsimultaneously. For example, isoprene can be directly condensed from theoff-gas stream to form a liquid. In some embodiments, the removal ofisoprene vapor from the fermentation off-gas and the conversion ofisoprene to a liquid phase are performed sequentially. For example,isoprene may be adsorbed to a solid phase and then extracted from thesolid phase with a solvent. In one embodiment, the isoprene is recoveredby using absorption stripping as described in U.S. Provisional Appl. No.61/288,142.

In some embodiments, any of the methods described herein further includepurifying the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be purified using standardtechniques. Purification refers to a process through which isoprene isseparated from one or more components that are present when the isopreneis produced. In some embodiments, the isoprene is obtained as asubstantially pure liquid. Examples of purification methods include (i)distillation from a solution in a liquid extractant and (ii)chromatography. As used herein, “purified isoprene” means isoprene thathas been separated from one or more components that are present when theisoprene is produced. In some embodiments, the isoprene is at leastabout 20%, by weight, free from other components that are present whenthe isoprene is produced. In various embodiments, the isoprene is atleast or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,by weight, pure. Purity can be assayed by any appropriate method, e.g.,by column chromatography, HPLC analysis, or GC-MS analysis.

In some embodiments, at least a portion of the gas phase remaining afterone or more recovery steps for the removal of isoprene is recycled byintroducing the gas phase into a cell culture system (such as afermentor) for the production of isoprene.

Isoprene Polymerization

The biosoprene compositions described herein can be subjected tochemical reactions to polymerize it to various products, such ascopolymers or polymers of specific molecular weight. As described above,“copolymers” refers to a polymer that is made from polymerizing isoprenewith another non-isoprene molecules, including but not limited to,1,3-butadiene, styrene, α-methyl styrene. Isoprene can be purified fromthe bioisoprene compositions prior to any polymerization reactions. Inone embodiment, the isoprene used is an isoprene monomer. In anotherembodiment, the isoprene used is polyisoprene in which isoprene monomershave been polymerized to form a polymer of isoprene units. In oneembodiment, the polyisoprene is not a linear polyisoprene (i.e.,non-linear polyisoprene). In another embodiment, the polyisoprene is alinear polyisoprene.

Polymers, either polyisoprene or copolymer, can also be either solublepolymer or gel polymer or a combination thereof. In one embodiment, apolymer is at least about 30% soluble polymer, at least about 40%soluble polymer, at least about 50% soluble polymer, at least about 60%soluble polymer, at least about 70% soluble polymer, at least about 80%soluble polymer, at least about 90% soluble polymer, at least about 95%soluble polymer, or at least about 100% soluble polymer, with theremainder being gel polymer.

In some embodiments, the polymer is a soluble polymer. The solublepolymer can have a molecular weight ranging from 300,000 to 800,000. Thesoluble polymer can have a molecular weight of at least about 300,000;400,000; 500,000, 600,000; 700,000; 800,000; 900,000; or 1,000,000. Thesoluble polymer can have a molecular weight of at most about 300,000;400,000; 500,000, 600,000; 700,000; 800,000; 900,000; or 1,000,000. Thesoluble polymer can be two dimensional.

In other embodiments, the polymer is a gel polymer. In one embodiment,the gel polymer has a molecular weight ranging from at least about 1million to at least about 50 million. In some embodiments, the gelpolymer is at least about 1 million, at least about 2 millions, at leastabout 3 millions, at least about 4 millions, at least about 5 millions,at least about 6 millions, at least about 7 millions, at least about 8millions, at least about 9 millions, at least about 10 millions, atleast about 15 millions, at least about 20 millions, at least about 25millions, at least about 30 millions, at least about 35 millions, atleast about 40 millions, at least about 45 millions, or at least about50 millions. In yet other embodiments, the gel polymer is at most about1 million, at most about 2 millions, at most about 3 millions, at mostabout 4 millions, at most about 5 millions, at most about 6 millions, atmost about 7 millions, at most about 8 millions, at most about 9millions, at most about 10 millions, at most about 15 millions, at mostabout 20 millions, at most about 25 millions, at most about 30 millions,at most about 35 millions, at most about 40 millions, at most about 45millions, or at least most about 50 millions. The gel component can bethree-dimensional.

In some aspects, polyisoprene polymers and methods of makingpolyisoprene polymers are provided. The polyisoprene may comprise one ormore of the embodiments described herein (e.g., an indicated δ¹³Cvalue). In some embodiments, any of the methods described herein (e.g.,methods of making and/or purifying isoprene) further includepolymerizing the isoprene (e.g., any isoprene described herein). Forexample, standard methods can be used to polymerize the purifiedisoprene to form cis-polyisoprene or other down stream products usingstandard methods. Accordingly, as described herein, in one aspect isprovided a tire comprising polyisoprene, such as cis-1,4-polyisopreneand/or trans-1,4-polyisoprene made from any of the isoprene compositionsdisclosed herein and/or using any of the methods of polymerizationdisclosed herein. In some of these embodiments, the polyisoprene (e.g.,any polyisoprene polymer described herein) is made from any isoprene orisoprene composition described herein.

In some aspects, the invention provides for systems for producing apolymer of isoprene comprising: (a) an isoprene starting compositionderived from renewable resources; and (b) a polymer produced from atleast a portion of the isoprene starting material; wherein at least aportion of the isoprene starting composition undergoes polymerizationwith other isoprene molecules to produce a polymer of isoprene with amolecular weight of about 5,000 to about 100,000. As used herein, “atleast a portion of the isoprene starting composition” can refer to atleast about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% of the isoprenestarting composition undergoing polymerization.

In other aspects, polyisoprene polymers and copolymers and methods ofmaking these types of polymers of various molecular weights areprovided. In one embodiment, the polymers have a molecular weight ofabout 5,000 to about 100,000. In other embodiments, the polymers have amolecular weight of at least about 6,000; 7,000; 8,000; 9,000; 10,000;12,000, 15,000; 20,000; 25,000; 30,000; 35,000; 40,000; 45,000; 50,000;55,000; 60,000; 70,000; 80,000, 90,000 or 100,000. In other embodiments,the polymers have a molecular weight of at most about 6,000; 7,000;8,000; 9,000; 10,000; 12,000, 15,000; 20,000; 25,000; 30,000; 35,000;40,000; 45,000; 50,000; 55,000; 60,000; 70,000; 80,000, 90,000 or100,000.

Additional methods and compositions are described in U.S. Provisionalpatent application No. 61/097,186, filed on Sep. 15, 2008, WO2010/031062, U.S. Provisional patent application No. 61/097,189, filedon Sep. 15, 2008, WO 2010/031077, U.S. Provisional patent applicationNo. 61/097,163, filed on Sep. 15, 2008, WO 2010/031079, and U.S. patentapplication Ser. No. 12/335,071 (US 2009/0203102 A1) all of which areincorporated by reference in their entireties, particularly with respectto compositions and methods for producing isoprene.

In one aspect, provided is a method for producing a polymer of isoprenederived from renewable resources comprising: (a) obtaining isoprene fromrenewable resources; (b) polymerizing isoprene derived from renewableresources; and (c) recovering the polymer produced. In some embodiments,the isoprene from renewable resources is obtained by a method whichcomprises the steps of (i) culturing cells comprising a heterologousnucleic acid encoding an isoprene synthase polypeptide under suitableculture conditions for the production of the isoprene, (ii) producingthe isoprene, and (iii) recovering the isoprene from the culture. Apolymer of isoprene derived from renewable resources, such as apolyisoprene homopolymer, a liquid polyisoprene polymer or a co-polymerof isoprene and one or more additional monomers, produced by any of themethods described herein is intended by the invention.

In some embodiments, the invention provide for a system for producing acopolymer of isoprene comprising: (a) an isoprene starting compositionderived from renewable resources; and (b) a polymer produced from atleast a portion of the isoprene starting material; wherein at least aportion of the isoprene starting composition undergoes polymerizationwith another non-isoprene molecule to produce a copolymer. As usedherein, “at least a portion of the isoprene starting composition” canrefer to at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% of theisoprene starting composition undergoing polymerization.

In some embodiments, the isoprene of this invention can be polymerizedinto useful polymers, including synthetic rubber, utilizing the sametechniques that are applicable to isoprene that is derived frompetrochemical sources. The polymerization and recovery of such isoprenecontaining polymers are suitably carried out according to variousmethods suitable for diene monomer polymerization processes. Thisincludes batchwise, semi-continuous, or continuous operations underconditions that exclude air and other atmospheric impurities,particularly oxygen and moisture. The polymerization of the isoprenemonomer may also be carried out in a number of different polymerizationreactor systems, including but not limited to bulk polymerization, vaporphase polymerization, solution polymerization, suspensionpolymerization, emulsion polymerization, and precipitationpolymerization systems. The commercially preferred methods ofpolymerization are typically solution polymerization and emulsionpolymerization.

In some embodiments, the system and compositions for producing a polymerof isoprene by polymerizing isoprene derived from renewable resourcesfurther comprises a catalyst for polymerizing isoprene. In someembodiments, the system and compositions further comprises apolymerization initiator. The polymerization reaction can also beinitiated using a vast array of different polymerization initiators orcatalyst systems. The initiator or catalyst system used will bedependent upon the desired characteristics of the isoprene containingpolymer being synthesized. For instance, in cases wherecis-1,4-polyisoprene rubber is being made a Ziegler Natta catalystsystem which is comprised of titanium tetrachloride and triethylaluminum can be utilized. In synthesizing other types of isoprenecontaining polymers other types of initiator systems may be needed. Forinstance, isoprene containing polymers can be made using a free radicalinitiator, a redox initiator, an anionic initiator, or a cationicinitiator. The preferred initiation or catalyst system will depend uponthe polymer microstructure, molecular weight, molecular weightdistribution, and chain branching desired. The preferred initiators willalso depend upon whether the isoprene is being homopolymerized orcopolymerized with additional monomers. In the case of copolymers theinitiator used will also depend upon whether it is desirable for thepolymer being made to have a random, non-random, or tapered distributionof repeat units that are derived of the particular monomers. Forinstance, anionic initiators or controlled free radical initiators aretypically used in synthesizing block copolymers having isoprene blocks.

It is important for the initiator or catalyst system employed to becompatible with the type of polymerization system used. For instance, inemulsion polymerizations free radical initiators are typically utilized.In solution polymerizations anionic initiators, such as alkyl lithiumcompounds, are typically employed to initiate the polymerization. Anadvantage of free radical polymerization is that reactions can typicallybe carried out under less rigorous conditions than ionicpolymerizations. Free radical initiation systems also exhibit a greatertolerance of trace impurities.

Conventional emulsion recipes may also be employed in polymerizingisoprene in accordance with the present invention; however, somerestrictions and modifications may arise either from the inclusion ofadditional comonomers, or the restrictions on polymerization parameters.In some embodiments, the system and compositions for producing a polymerof isoprene by polymerizing isoprene derived from renewable resourcesfurther comprises an ionic surfactant. Ionic surfactants, known in theart, including sulfonate detergents and carboxylate, sulfate, andphosphate soaps are useful in this invention. The level of ionicsurfactant is computed based upon the total weight of the organiccomponents and may range from about 2 to 30 parts by weight of ionicsurfactant per 100 parts by weight of organic components.

Examples of free radical initiators that are useful in the practice ofthe present invention are those known as “redox” initiators, such ascombinations of chelated iron salts, sodium formaldehyde sulfoxylate,and organic hydroperoxides. Representative of organic hydroperoxides arecumene hydroperoxide, paramenthane hydroperoxide, and tertiary butylhydroperoxide. Tertiary butyl hydroperoxide (t-BHP), tertiary butylperacetate (t-BPA) and “azo” initiators, such as azobisiobutyronitrile(AIBN), are preferred.

The reaction temperature utilized in free radical polymerizations istypically maintained in the range of 0° C. to 150° C. Temperaturesbetween about 20° C. and 120° C. are generally preferred andtemperatures within the range of 60° C. to 100° C. are normally mostpreferred. The reaction pressure is not critical. It is typically onlysufficiently high to maintain liquid phase reaction conditions; it maybe autogenic pressure, which will vary depending upon the components ofthe reaction mixture and the temperature, or it may be higher, e.g., upto 1000 psi.

In some embodiments, the method for producing a polymer of isoprenederived from renewable resources comprises polymerizing isoprene derivedfrom renewable resources in a batch process. In batch operations, thepolymerization time can be varied as desired from as little as a fewminutes to as lone as several days. Polymerization in batch processesmay be terminated when monomer is no longer absorbed, or earlier, ifdesired, e.g., if the reaction mixture becomes too viscous. Incontinuous operations, the polymerization mixture may be passed througha reactor or series of reactors of any suitable design. Thepolymerization reactions in such cases are suitably adjusted by varyingthe residence time in the reactor system. Residence times vary with thetype of reactor system and range from 10 to 15 minutes to 24 or morehours. The concentration of monomer in the reaction mixture may varyupwards from 5 percent by weight of the reaction mixture, depending onthe conditions employed; the range from 20 to 80 percent by weight ispreferred.

In some embodiments, the system and compositions for producing a polymerof isoprene by polymerizing isoprene derived from renewable resourcesfurther comprises a suitable organic solvent. In some embodiments, thepolymerization of isoprene is carried out in a suitable organic solventthat is liquid under the conditions of reaction and which is relativelyinert. The solvent may have the same number of carbon atoms per moleculeas the diene reactant or it may be in a different boiling range.Preferred organic solvents are normally alkanes and cycloalkanes. Thesolvents can be comprised of one or more aromatic, paraffinic orcycloparaffinic compounds. These solvents will normally contain fromabout 4 carbon atoms per mole to about 10 carbon atoms per molecule andwill be liquid under the conditions of the polymerization. Somerepresentative examples of suitable organic solvents include pentane,isooctane, cyclohexane, methylcyclohexane, isohexane, n-heptane,n-octane, n-hexane, benzene, toluene, xylene, ethylbenzene,diethylbenzene, isobutylbenzene, petroleum ether, kerosene, petroleumspirits, petroleum naphtha, and the like, alone or in admixture.Aromatic hydrocarbons, such as benzene, toluene, isopropylbenzene,xylene, or halogenated aromatic compounds, such as chlorobenzene,bromobenzene, or orthodichlorobenzene, may also be employed, but are notpreferred in most cases. Other useful solvents include tetrahydrofuranand dioxane.

In the solution polymerization, there will normally be from 5 to 30weight percent monomers in the polymerization medium. Suchpolymerization media are, of course, comprised of the organic solventand monomers. In most cases, it will be preferred for the polymerizationmedium to contain from 10 to 25 weight percent monomers. It is generallymore preferred for the polymerization medium to contain 15 to 20 weightpercent monomers.

The polymerization is typically carried out to attain an essentiallycomplete conversion of monomers into polymer. Incremental monomeraddition, or a chain transfer agent, may be used in order to avoidexcessive gel formation. Such minor modifications are within the skillof the artisan. After the polymerization is complete, the polymer isrecovered from a slurry or solution of the polymer. A simple filtrationmay be adequate to separate polymer from diluent. Other means forseparating polymer from diluent may be employed. The polymer may betreated, separately or while slurried in the reaction mixture, in orderto separate residues. Such treatment may be with alcohols such asmethanol, ethanol, or isopropanol, with acidified alcohols, or withother similar polar liquids. In many cases the polymers are obtained inhydrocarbon solutions and the polymer can be recovered by coagulationwith acidified alcohol, e.g., rapidly stirred methanol or isopropanolcontaining 2% hydrochloric acid. Following this initial coagulation, thepolymers may be washed with an appropriate liquid, such as methanol.

In some embodiments, the system and compositions for producing a polymerof isoprene by polymerizing isoprene derived from renewable resourcesfurther comprises one or more additional monomers. As has beenpreviously noted, the isoprene can also be copolymerized with one ormore additional comonomers to make useful copolymers. Some adjustmentsin the polymerization recipe or reaction conditions may be necessary toobtain a satisfactory rate of polymer formation, depending on therelative amount of isoprene included and the other monomers involved.Examples of comonomers that are useful in the practice of this inventioninclude other diene monomers, such as 1,3-butadiene and hexadienes.Vinyl aromatic monomers can also be copolymerizable with isoprene tomake useful polymers. Such vinyl aromatic monomers include styrene,α-methylstyrene, divinylbenzene, vinyl chloride, vinyl acetate,vinylidene chloride, methyl methacrylate, ethyl acrylate, vinylpyridine,acrylonitrile, methacrylonitrile, methacrylic acid, itaconic acid andacrylic acid. Mixtures of different comonomers can also be employed atdiffering levels.

In some embodiments, the isoprene monomer is copolymerized with one ormore additional conjugated diolefin monomers. Those containing from 4 to8 carbon atoms are generally preferred for commercial purposes. Somespecific representative examples of conjugated diolefin monomers thatcan be copolymerized with isoprene include 1,3-butadiene,2,3-dimethyl-1,3-butadiene, piperylene, 3-butyl-1,3-octadiene,2-phenyl-1,3-butadiene, and the like, alone or in admixture.

In some embodiments, the isoprene monomer is copolymerized with one ormore additional ethylenically unsaturated monomers. Some representativeexamples of ethylenically unsaturated monomers that can copolymerizedwith isoprene include alkyl acrylates, such as methyl acrylate, ethylacrylate, butyl acrylate, methyl methacrylate and the like; vinylidenemonomers having one or more terminal CH₂═CH— groups; vinyl aromaticssuch as styrene, α-methylstyrene, bromostyrene, chlorostyrene,fluorostyrene and the like; α-olefins such as ethylene, propylene,1-butene and the like; vinyl halides, such as vinylbromide, chloroethene(vinylchloride), vinylfluoride, vinyliodide, 1,2-dibromoethene,1,1-dichloroethene (vinylidene chloride), 1,2-dichloroethene and thelike; vinyl esters, such as vinyl acetate; α,β-olefinically unsaturatednitriles, such as acrylonitrile and methacrylonitrile; α,β-olefinicallyunsaturated amides, such as acrylamide, N-methylacrylamide,N,N-dimethylacrylamide, methacrylamide and the like. Functionalizedmonomers can also optionally be copolymerized with the isoprene inmaking useful rubbery polymers. Functionalized monomers of this type andmethods by which they can be incorporated into rubbery polymers aredescribed in U.S. Pat. No. 6,627,721 and U.S. Pat. No. 6,936,669. Theteachings of U.S. Pat. No. 6,627,721 and U.S. Pat. No. 6,936,669 areincorporated herein by reference for the purpose of describing suchfunctionalized monomers and their incorporation into isoprene containingpolymers.

Rubbery polymers which are copolymers of one or more diene monomers withone or more other ethylenically unsaturated monomers will normallycontain from about 50 weight percent to about 99 weight percentconjugated diolefin monomers (including isoprene) and from about 1weight percent to about 50 weight percent of the other ethylenicallyunsaturated monomers in addition to the conjugated diolefin monomers.For example, rubbery copolymers of isoprene monomer with vinylaromaticmonomers, such as styrene-isoprene rubbers will normally which containfrom 50 to 95 weight percent isoprene and from 5 to 50 weight percentvinylaromatic monomers.

Vinyl aromatic monomers are probably the most important group ofethylenically unsaturated monomers which are commonly incorporated intoisoprene containing rubbers. Such vinyl aromatic monomers typicallycontain from 8 to 20 carbon atoms. Usually, the vinyl aromatic monomerwill contain from 8 to 14 carbon atoms. The most widely used vinylaromatic monomer is styrene. Some examples of vinyl aromatic monomersthat can be utilized include styrene, 1-vinylnaphthalene,2-vinylnaphthalene, α-methylstyrene, 4-phenylstyrene, 3-methylstyreneand the like.

Some representative examples of isoprene containing rubbery polymersinclude cis-1,3-polyisoprene homopolymer rubber, 3,4-polyisoprenerubber, styrene-isoprene rubber (SIR), β-methylstyrene-isoprene rubber,styrene-isoprene-butadiene rubber (SIBR), styrene-isoprene rubber (SIR),isoprene-butadiene rubber (IBR), α-methylstyrene-isoprene-butadienerubber and α-methylstyrene-styrene-isoprene-butadiene rubber. In caseswhere the rubbery polymer is comprised of repeat units that are derivedfrom two or more monomers, the repeat units which are derived from thedifferent monomers, including the isoprene, will normally be distributedin an essentially random manner. The repeat units that are derived fromthe monomers differ from the monomer in that a double bond is normallyconsumed in by the polymerization reaction.

In some embodiments, the method for producing a polymer of isoprenederived from renewable resources comprises polymerizing isoprene derivedfrom renewable resources in a continuous process. The rubbery polymercan be made by solution polymerization in a batch process or in acontinuous process by continuously charging the isoprene monomer andoptionally additional monomers into a polymerization zone. Thepolymerization zone will typically be a polymerization reactor or aseries of polymerization reactors. The polymerization zone will normallyprovide agitation to keep the monomers, polymer, initiator, and modifierwell dispersed throughout the organic solvent the polymerization zone.Such continuous polymerizations are typically conducted in a multiplereactor system. The rubbery polymer synthesized is continuouslywithdrawn from the polymerization zone. The monomer conversion attainedin the polymerization zone will normally be at least about 85 percent.It is preferred for the monomer conversion to be at least about 90percent.

In some embodiments, the system and compositions for producing a polymerof isoprene by polymerizing isoprene derived from renewable resourcesfurther comprises a polymerization initiator and a polar modifier. Thepolymerization can be initiated with an anionic initiator, such as analkyl lithium compound. The alkyl lithium compounds that can be usedwill typically contain from 1 to about 8 carbon atoms, such as n-butyllithium. The amount of the lithium initiator utilized will vary with themonomers being polymerized and with the molecular weight that is desiredfor the polymer being synthesized. However, as a general rule, from 0.01to 1 phm (parts per 100 parts by weight of monomer) of the lithiuminitiator will be utilized. In most cases, from 0.01 to 0.1 phm of thelithium initiator will be utilized with it being preferred to utilize0.025 to 0.07 phm of the lithium initiator.

Such anionic polymerizations are optionally conducted in the presence ofpolar modifiers, such as alkyltetrahydrofurfuryl ethers. Somerepresentative examples of specific polar modifiers that can be usedinclude methyltetrahydrofurfuryl ether, ethyltetrahydrofurfuryl ether,propyltetrahydrofurfuryl ether, butyltetrahydrofurfuryl ether,hexyltetrahydrofurfuryl ether, octyltetrahydrofurfuryl ether,dodecyltetrahydrofurfuryl ether, diethyl ether, di-n-propyl ether,diisopropyl ether, di-n-butyl ether, tetrahydrofuran, dioxane, ethyleneglycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycoldimethyl ether, diethylene glycol diethyl ether, triethylene glycoldimethyl ether, trimethylamine, triethylamine,N,N,N′,N′-tetramethylethylenediamine, N-methylmorpholine,N-ethylmorpholine, or N-phenylmorpholine.

The polar modifier will typically be employed at a level wherein themolar ratio of the polar modifier to the lithium initiator is within therange of about 0.01:1 to about 5:1. The molar ratio of the polarmodifier to the lithium initiator will more typically be within therange of about 0.1:1 to about 4:1. It is generally preferred for themolar ratio of polar modifier to the lithium initiator to be within therange of about 0.25:1 to about 3:1. It is generally most preferred forthe molar ratio of polar modifier to the lithium initiator to be withinthe range of about 0.5:1 to about 3:2.

The polymerization temperature utilized in such anionic polymerizationscan vary over a broad range of from about −20° C. to about 180° C. Inmost cases, a polymerization temperature within the range of about 30°C. to about 125° C. will be utilized. It is typically preferred for thepolymerization temperature to be within the range of about 45° C. toabout 100° C. It is typically most preferred for the polymerizationtemperature to be within the range of about 60° C. to about 90° C. Thepressure used will normally be sufficient to maintain a substantiallyliquid phase under the conditions of the polymerization reaction.

In some embodiments, the system and compositions for producing a polymerof isoprene by polymerizing isoprene derived from renewable resourcesfurther comprises a polymerization chain terminator such as an alcohol,a terminating agent, or a coupling agent. Such anionic polymerizationsof isoprene are normally conducted for a length of time sufficient topermit substantially complete polymerization of the isoprene and anyadditional monomers that are present. In other words, the polymerizationis normally carried out until high conversions of at least about 85percent are attained. The polymerization is then normally terminated bythe addition of an agent, such as an alcohol, a terminating agent, or acoupling agent. For example, a tin halide and/or silicon halide can beused as a coupling agent. The tin halide and/or the silicon halide arecontinuously added in cases where asymmetrical coupling is desired. Thiscontinuous addition of tin coupling agent and/or the silicon couplingagent is normally done in a reaction zone separate from the zone wherethe bulk of the polymerization is occurring. The coupling agents willnormally be added in a separate reaction vessel after the desired degreeof conversion has been attained. The coupling agents can be added in ahydrocarbon solution, e.g., in cyclohexane, to the polymerizationadmixture with suitable mixing for distribution and reaction. In otherwords, the coupling will typically be added only after a high degree ofconversion has already been attained. For instance, the coupling agentwill normally be added only after a monomer conversion of greater thanabout 85 percent has been realized. It will typically be preferred forthe monomer conversion to reach at least about 90 percent before thecoupling agent is added.

The tin halides used as coupling agents will normally be tintetrahalides, such as tin tetrachloride, tin tetrabromide, tintetrafluoride or tin tetraiodide. However, tin trihalides can alsooptionally be used. Polymers coupled with tin trihalides having amaximum of three arms. This is, of course, in contrast to polymerscoupled with tin tetrahalides which have a maximum of four arms. Toinduce a higher level of branching, tin tetrahalides are normallypreferred. As a general rule, tin tetrachloride is most preferred.

The silicon coupling agents that can be used will normally be silicontetrahalides, such as silicon tetrachloride, silicon tetrabromide,silicon tetrafluoride or silicon tetraiodide. However, silicontrihalides can also optionally be used. Polymers coupled with silicontrihalides having a maximum of three arms. This is, of course, incontrast to polymers coupled with silicon tetrahalides which have amaximum of four arms. To induce a higher level of branching, silicontetrahalides are normally preferred. As a general rule, silicontetrachloride is most preferred of the silicon coupling agents.

A combination of a tin halide and a silicon halide can optionally beused to couple the rubbery polymer. By using such a combination of tinand silicon coupling agents improved properties for tire rubbers, suchas lower hysteresis, can be attained. It is particularly desirable toutilize a combination of tin and silicon coupling agents in tire treadcompounds that contain both silica and carbon black. In such cases, themolar ratio of the tin halide to the silicon halide employed in couplingthe rubbery polymer will normally be within the range, of 20:80 to 95:5.The molar ratio of the tin halide to the silicon halide employed incoupling the rubbery polymer will more typically be within the range of40:60 to 90:10. The molar ratio of the tin halide to the silicon halideemployed in coupling the rubbery polymer will preferably be within therange of 60:40 to 85:15. The molar ratio of the tin halide to thesilicon halide employed in coupling the rubbery polymer will mostpreferably be within the range of 65:35 to 80:20.

Broadly, and exemplary, a range of about 0.01 to 4.5 milliequivalents oftin coupling agent (tin halide and silicon halide) is employed per 100grams of the rubbery polymer. It is normally preferred to utilize about0.01 to about 1.5 milliequivalents of the coupling agent per 100 gramsof polymer to obtain the desired Mooney viscosity. The larger quantitiestend to result in production of polymers containing terminally reactivegroups or insufficient coupling. One equivalent of tin coupling agentper equivalent of lithium is considered an optimum amount for maximumbranching. For instance, if a mixture tin tetrahalide and silicontetrahalide is used as the coupling agent, one mole of the couplingagent would be utilized per four moles of live lithium ends. In caseswhere a mixture of tin trihalide and silicon trihalide is used as thecoupling agent, one mole of the coupling agent will optimally beutilized for every three moles of live lithium ends. The coupling agentcan be added in a hydrocarbon solution, e.g., in cyclohexane, to thepolymerization admixture in the reactor with suitable mixing fordistribution and reaction.

After the coupling has been completed, a tertiary chelating alkyl1,2-ethylenediamine or a metal salt of a cyclic alcohol can optionallybe added to the polymer cement to stabilize the coupled rubbery polymer.In most cases, from about 0.01 phr (parts by weight per 100 parts byweight of dry rubber) to about 2 phr of the chelating alkyl1,2-ethylenediamine or metal salt of the cyclic alcohol will be added tothe polymer cement to stabilize the rubbery polymer. Typically, fromabout 0.05 phr to about 1 phr of the chelating alkyl 1,2-ethylenediamineor metal salt of the cyclic alcohol will be added. More typically, fromabout 0.1 phr to about 0.6 phr of the chelating alkyl1,2-ethylenediamine or the metal salt of the cyclic alcohol will beadded to the polymer cement to stabilize the rubbery polymer.

The terminating agents that can be used to stop the polymerization andto “terminate” the living rubbery polymer include tin monohalides,silicon monohalides, N,N,N′,N′-tetradialkyldiamino-benzophenones (suchas tetramethyldiaminobenzophenone and the like),N,N-dialkylamino-benzaldehydes (such as dimethylaminobenzaldehyde andthe like), 1,3-dialkyl-2-imidazolidinones (such as1,3-dimethyl-2-imidazolidinone and the like), 1-alkyl substitutedpyrrolidinones; 1-aryl substituted pyrrolidinones,dialkyl-dicycloalkyl-carbodiimides containing from about 5 to about 20carbon atoms, and dicycloalkyl-carbodiimides containing from about 5 toabout 20 carbon atoms.

After the termination step, and optionally the stabilization step, hasbeen completed, the rubbery polymer can be recovered from the organicsolvent. The coupled rubbery polymer can be recovered from the organicsolvent and residue by means such as chemical (alcohol) coagulation,thermal desolventization, or other suitable method. For instance, it isoften desirable to precipitate the rubbery polymer from the organicsolvent by the addition of lower alcohols containing from about 1 toabout 4 carbon atoms to the polymer solution. Suitable lower alcoholsfor precipitation of the rubber from the polymer cement includemethanol, ethanol, isopropyl alcohol, normal-propyl alcohol and t-butylalcohol. The utilization of lower alcohols to precipitate the rubberypolymer from the polymer cement also “terminates” any remaining livingpolymer by inactivating lithium end groups. After the coupled rubberypolymer is recovered from the solution, steam-stripping can be employedto reduce the level of volatile organic compounds in the coupled rubberypolymer. Additionally, the organic solvent can be removed from therubbery polymer by drum drying, extruder drying, vacuum drying, and thelike.

As has previously been explained, synthetic cis-1,3-polyisoprene rubberthat is similar enough to allow for free substitution with naturalrubber can be produced by the solution polymerization of isoprene with aZiegler Natta catalyst system that is comprised of titaniumtetrachloride (TiCl₄) and an organoaluminum compound, such as triethylaluminum, Al—(CH₂—CH₃)₃. The polyisoprene rubber that is made with thisZiegler Natta catalyst system has a high cis-microstructure contain ofup to 98 percent that closely assimilates that of natural rubber fromHevea Brasiliensis (the common rubber tree) which has acic-microstructure content of virtually 100 percent. However, thisslight difference in polymer microstructure results of physicalproperties that are inferior to those of natural rubber is certainrespects. For instance, natural rubber typically exhibits green strengththat is superior to that of synthetic cis-1,4-polyisoprene rubber. Onthe other hand, in certain other respects synthetic cis-1,4-polyisoprenerubber is superior to natural rubber from the Hevea Brasiliensis,guayule, and Taraxacum kok-Saghyz (Russian dandelion). For instance,natural rubber contains residual proteins, soaps, resins, and sugarssince it comes from plants. The presence of these residual impuritiescan be extremely detrimental in some applications. For instance, thepresence of residual proteins in rubber products can cause seriousallergic reactions in some people and are a major concern formanufacturers of some rubber-containing products, such as rubber gloves,condoms, syringe plungers, and the like. In any case, the syntheticpolyisoprene homopolymer rubbers of this invention that are free fromproteins, soaps, resins, and sugars present in natural rubber, includingnatural rubber from the Hevea Brasiliensis.

U.S. Pat. No. 3,931,136 discloses a process for producing high molecularweight cis-1,4-polyisoprene. The catalyst used in this process is athree-component mixture of (A) a titanium tetrachloride, (B) anorganoaluminum compound of the formula AlR₃, where each R represents analkyl group, preferably an alkyl group containing 1 to 8 carbon atoms,an aryl group, preferably a phenyl group, or a cycloalkyl group,preferably a cyclohexyl group, and (C) a beta-diketone of the formula:

where R′ and R″ can be the same or different and represent an alkylgroup or a aryl group. R′ and R″ will preferably represent an alkylgroup containing from 1 to 5 carbon atoms or a phenyl group. Theteachings of U.S. Pat. No. 3,931,136 are incorporated herein byreference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizingcis-1,4-polyisoprene.

A solution polymerization technique for synthesizingcis-1,4-polyisoprene with a catalyst system that is comprised of amixture of titanium tetrachloride and a trialkylaluminum compound isdisclosed by U.S. Pat. No. 4,430,487. In this process the polymerizationis shortstopped with 4,7-diaza-decane-1,10-diamine. The teachings ofU.S. Pat. No. 4,430,487 are incorporated herein by reference for thepurpose of teaching catalyst systems and polymerization techniques thatcan be used in synthesizing cis-1,4-polyisoprene.

The synthesis of cis-1,4-polyisoprene by polymerizing isoprene with acatalyst system which is comprised of a titanium tetrahalide, atrialkylaluminum compound and diphenylether can result in the formationof unwanted gel. U.S. Pat. No. 5,919,876 discloses that gel formationcan be reduced by conducting such polymerizations in the presence of adiarylamine, such as para-styrenated diphenylamine. U.S. Pat. No.5,919,876 more specifically discloses a process for synthesizingcis-1,4-polyisoprene having a low gel content which comprisespolymerizing isoprene in an inert organic solvent with a preformedcatalyst system which is made by reacting an organoaluminum compoundwith titanium tetrahalide, such as titanium tetrachloride, in thepresence of at least one ether, wherein said polymerization is conductedat a temperature which is within the range of about 0° C. to about 100°C., and wherein said polymerization is conducted in the presence of adiarylamine. The teachings of U.S. Pat. No. 5,919,867 are incorporatedherein by reference for the purpose of teaching catalyst systems andsolution polymerization techniques that can be used in synthesizingcis-1,4-polyisoprene rubber.

Cis-1,4-polyisoprene can be made by vapor phase polymerization utilizinga preformed catalyst that is made by reacting an organoaluminum compoundwith titanium tetrachloride. U.S. Pat. No. 6,066,705 discloses a methodfor vapor phase polymerizing isoprene into cis-1,4-polyisoprene in aprocess comprising the steps of: (1) charging into a reaction zone saidisoprene and a preformed catalyst system which is made by reacting anorganoaluminum compound with titanium tetrachloride, preferably in thepresence of at least one ether; wherein the isoprene is maintained inthe vapor phase in said reaction zone by a suitable combination oftemperature and pressure; (2) allowing said isoprene to polymerize intocis-1,4-polyisoprene at a temperature within the range of about 35° C.to about 70° C.; and (3) withdrawing said cis-1,4-polyisoprene from saidreaction zone. It has been determined that gel formation can be reducedin such vapor phase polymerizations by conducting the polymerization ofthe isoprene monomer in the presence of a diarylamine, such aspara-styrenated diphenylamine. The teachings of U.S. Pat. No. 6,066,705are incorporated herein by reference for the purpose of teachingcatalyst systems and vapor phase polymerization techniques that can beused in synthesizing cis-1,4-polyisoprene rubber.

Polyisoprene rubber that is clear (transparent) and of high purity canbe synthesized utilizing a neodymium catalyst system. U.S. Pat. No.6,780,948 relates to such a process for the synthesis of polyisoprenerubber which comprises polymerizing isoprene monomer in the presence ofa neodymium catalyst system, wherein the neodymium catalyst system isprepared by (1) reacting a neodymium carboxylate with an organoaluminumcompound in the presence of isoprene for a period of about 10 minutes toabout 30 minutes to produce neodymium-aluminum catalyst component, and(2) subsequently reacting the neodymium-aluminum catalyst component witha dialkyl aluminum chloride for a period of at least 30 minutes toproduce the neodymium catalyst system. The teachings of U.S. Pat. No.5,919,867 are incorporated herein by reference for the purpose ofteaching catalyst systems and polymerization techniques that can be usedin synthesizing cis-1,4-polyisoprene rubber that is of high purity.

U.S. Pat. No. 7,091,150 and U.S. Pat. No. 7,199,201 disclose the use ofa neodymium catalyst system to polymerize isoprene monomer intosynthetic polyisoprene rubber having an extremely highcis-microstructure content and high stereo regularity. This polyisoprenerubber will crystallize under strain and can be compounded into rubberformulations in a manner similar to natural rubber. This technique morespecifically discloses a process for the synthesis of polyisoprenerubber which comprises polymerizing isoprene monomer in the presence ofa neodymium catalyst system, wherein the neodymium catalyst system isprepared by a process that comprises (1) reacting a neodymiumcarboxylate with an organoaluminum compound in an organic solvent toproduce neodymium-aluminum catalyst component, and (2) subsequentlyreacting the neodymium-aluminum catalyst component with an elementalhalogen to produce the neodymium catalyst system. In practicing thisprocess, the neodymium catalyst system is typically void ofnickel-containing compounds.

The synthetic polyisoprene rubber made by this process is comprised ofrepeat units that are derived from isoprene, wherein the syntheticpolyisoprene rubber has a cis-microstructure content which is within therange of 98.0% to 99.5%, a 3,4-microstructure content which is withinthe range of 0.5% to 2.0%, and a trans-microstructure content which iswithin the range of 0.0% to 0.5%. The teachings of U.S. Pat. No.7,091,150 and U.S. Pat. No. 7,199,201 are incorporated herein byreference for the purpose of teaching neodymium catalyst systems andpolymerization techniques that can be used in synthesizingcis-1,4-polyisoprene rubber of extremely high cis-microstructure contentand high stereo regularity.

Single component lanthanide catalysts, such as lanthanide diiodides, canalso be used in the synthesis of polyisoprene having extremely highcis-microstructure contents. For instance, thulium diiodide, dysprosiumdiiodide, and neodymium diiodide can initiate the polymerization ofisoprene into high cis-1,4-polyisoprene rubber without the need for anyadditional catalyst components. Lanthanide diiodides can accordingly beused to initiate the polymerization of isoprene monomer into highcis-1,4-polyisoprene under solution polymerization conditions.

U.S. Pat. No. 4,894,425 reveals a process for synthesizing polyisoprenethat may possess functional groups and that contains more than 70percent 1,2- and 3,4-structural units. This process involves the anionicpolymerization of isoprene in an inert hydrocarbon solvent in thepresence of an organolithium compound as the catalyst and an ether asthe cocatalyst, wherein the cocatalyst used is an ethylene glycoldialkyl ether of the formula R¹—O—CH₂—CH₂—O—R² wherein R¹ and R² arealkyl groups having different numbers of carbon atoms, selected from thegroup consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, and tert-butyl, and wherein the sum of the carbonatoms in the two alkyl groups R¹ and R² is within the range of 5 to 7.The teachings of U.S. Pat. No. 4,894,425 are incorporated herein byreference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizing polyisoprenehaving a high 1,2- and 3,4-microstructure content.

Crystallizable 3,4-polyisoprene can be synthesized in organic solventsto quantitative yields after short polymerization times by utilizing thecatalyst systems described by U.S. Pat. No. 5,082,906. The3,4-polyisoprene made utilizing this catalyst system is straincrystallizable and can be employed in tire treads which provide improvedtraction and improved cut growth resistance. U.S. Pat. No. 5,082,906specifically discloses a process for the synthesis of 3,4-polyisoprenewhich comprises polymerizing isoprene monomer in an organic solvent at atemperature which is within the range of about −10° C. to about 100° C.in the presence of a catalyst system which is composed of (a) anorganoiron compound, (b) an organoaluminum compound, (c) a chelatingaromatic amine, and (d) a protonic compound; wherein the molar ratio ofthe chelating amine to the organoiron compound is within the range ofabout 0.1:1 to about 1:1, wherein the molar ratio of the organoaluminumcompound to the organoiron compound is within the range of about 5:1 toabout 200:1, and wherein the molar ratio of the protonic compound to theorganoaluminum compound is within the range of about 0.001:1 to about0.2:1. The teachings of U.S. Pat. No. 5,082,906 are incorporated hereinby reference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizing polyisoprenehaving a high 3,4-microstructure content and which is straincrystallizable.

U.S. Pat. No. 5,356,997 also relates to a process for the synthesis ofstrain crystallizable 3,4-polyisoprene. This 3,4-polyisoprene has a3,4-microstructure content which is within the range of about 65% toabout 85%, a cis-1,4-microstructure content which is within the range ofabout 15% to about 35%, and essentially no trans-1,4-microstructure or1,2-microstructure. It can be synthesized in organic solvents toquantitative yields after short polymerization times. U.S. Pat. No.5,356,997 specifically discloses a process for the synthesis of3,4-polyisoprene which comprises polymerizing isoprene monomer in anorganic solvent at a temperature which is within the range of about −10°C. to about 100° C. in the presence of a catalyst system which iscomprised of (a) an organoiron compound which is soluble in the organicsolvent, wherein the iron in the organoiron compound is in the +3oxidation state, (b) a partially hydrolyzed organoaluminum compoundwhich was prepared by adding a protonic compound selected from the groupconsisting of water, alcohols and carboxylic acids to the organoaluminumcompound, and (c) a chelating aromatic amine; wherein the molar ratio ofthe chelating amine to the organoiron compound is within the range ofabout 0.1:1 to about 1:1, wherein the molar ratio of the organoaluminumcompound to the organoiron compound is within the range of about 5:1 toabout 200:1, and wherein the molar ratio of the protonic compound to theorganoaluminum compound is within the range of about 0.001:1 to about0.2:1. The teachings of U.S. Pat. No. 5,356,997 are incorporated hereinby reference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizing polyisoprenehaving a high 3,4-microstructure content and which is straincrystallizable.

U.S. Pat. No. 5,677,402 reveals a process for preparing 3,4-polyisoprenerubber which comprises polymerizing isoprene monomer with anorganolithium initiator at a temperature which is within the range ofabout 30° C. to about 100° C. in the presence of a sodium alkoxide and apolar modifier, wherein the molar ratio of the sodium alkoxide to theorganolithium initiator is within the range of about 0.05:1 to about3:1; and wherein the molar ratio of the polar modifier to theorganolithium initiator is within the range of about 0.25:1 to about5:1. The teachings of U.S. Pat. No. 5,677,402 are incorporated herein byreference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizing3,4-polyisoprene.

U.S. Pat. No. 7,351,768 discloses the synthesis of liquid polyisoprenehaving a weight average molecular weight which is within the range of5,000 to 100,000 and preferable within the range of 20,000 to 80,000.The teachings of U.S. Pat. No. 5,677,402 are incorporated herein byreference for the purpose illustrating the synthesis of liquidpolyisoprene.

U.S. Pat. No. 6,576,728 discloses a process for the copolymerization ofstyrene and isoprene to produce low vinyl styrene-isoprene rubber havinga random distribution of repeat units that are derived from styrene. Theinitiator systems employed in these polymerizations are comprised of (a)a lithium initiator and (b) a member selected from the group consistingof (1) a sodium alkoxide, (2) a sodium salt of a sulfonic acid, and (3)a sodium salt of a glycol ether. It is important for the initiatorsystem used in these polymerizations to be free of polar modifiers, suchas Lewis bases. The teachings of U.S. Pat. No. 6,576,728 areincorporated herein by reference for the purpose illustrating thesynthesis of styrene-isoprene rubber.

U.S. Pat. No. 6,313,216 discloses a process for synthesizing randomstyrene-isoprene rubber comprising: (1) continuously charging isoprene,styrene, an initiator, and a solvent into a first polymerization zone,(2) allowing the isoprene and styrene to copolymerize in the firstpolymerization zone to total conversion of 60 to 95 percent to produce apolymer cement containing living styrene-isoprene chains, (3)continuously charging the polymer cement containing livingstyrene-isoprene chains and additional isoprene monomer into a secondpolymerization zone, wherein from 5 to 40 percent of the total amount ofisoprene changed is charged into the second polymerization zone, (4)allowing the copolymerization to continue in the second polymerizationzone to a conversion of the isoprene monomer of at least 90 percentwherein the total conversion of styrene and isoprene in the secondpolymerization zone is limited to a maximum of 98 percent, (5)withdrawing a polymer cement of random styrene-isoprene rubber havingliving chain ends from the second reaction zone, (6) killing the livingchain ends on the random styrene-isoprene rubber, and (7) recovering therandom styrene-isoprene rubber from the polymer cement, wherein thecopolymerizations in the first polymerization zone and the secondpolymerization zone are carried out at a temperature which is within therange of 70° C. to 100° C., and wherein the amount of styrene chargedinto the first polymerization zone is at least 2 percent more than thetotal amount of styrene bound into the rubber. The teachings of U.S.Pat. No. 6,313,216 are incorporated herein by reference for the purposeillustrating the synthesis of styrene-isoprene rubber.

Isoprene-butadiene copolymers having high vinyl contents can besynthesized in organic solvents to high yields after shortpolymerization times by utilizing the process disclosed in U.S. Pat. No.5,061,765. The isoprene-butadiene copolymers made utilizing this processhave a glass transition temperature which is within the range of about0° C. to about −60° C. and can be employed in tire treads which provideimproved traction and improved cut growth resistance. U.S. Pat. No.5,061,765 more specifically discloses a process for the synthesis ofisoprene-butadiene copolymers having a high vinyl content whichcomprises copolymerizing isoprene monomer and butadiene monomer in anorganic solvent at a temperature which is within the range of about −10°C. to about 100° C. in the presence of a catalyst system which iscomprised of (a) an organoiron compound, (b) an organoaluminum compound,(c) a chelating aromatic amine, and (d) a protonic compound; wherein themolar ratio of the chelating amine to the organoiron compound is withinthe range of about 0.1:1 to about 1:1, wherein the molar ratio of theorganoaluminum compound to the organoiron compound is within the rangeof about 5:1 to about 200:1, and wherein the molar ratio of the protoniccompound to the organoaluminum compound is within the range of about0.001:1 to about 0.2:1. The teachings of U.S. Pat. No. 5,061,765 areincorporated herein by reference for the purpose illustrating thesynthesis of isoprene-butadiene rubber.

A technique for synthesizing rubbery terpolymers of styrene, isopreneand butadiene is disclosed in U.S. Pat. No. 5,137,998. These rubberyterpolymers exhibit an excellent combination of properties forutilization in tire tread rubber compounds. By utilizing suchterpolymers in tire treads, tires having improved wet skid resistancecan be built without sacrificing rolling resistance or tread wearcharacteristics. U.S. Pat. No. 5,137,998 more specifically discloses aprocess for preparing a rubbery terpolymer of styrene, isoprene, andbutadiene having multiple glass transition temperatures and having anexcellent combination of properties for use in making tire treads whichcomprises: terpolymerizing styrene, isoprene and 1,3-butadiene in anorganic solvent at a temperature of no more than about 40° C. in thepresence of (a) at least one member selected from the group consistingof tripiperidino phosphine oxide and alkali metal alkoxides and (b) anorganolithium compound. The teachings of U.S. Pat. No. 5,137,998 areincorporated herein by reference for the purpose illustrating thesynthesis of styrene-isoprene-butadiene rubber.

A liquid isoprene-butadiene rubber (IBR) which is particularly valuablefor use in making treads for high performance automobile tires,including race tires, that exhibit superior dry traction characteristicsand durability, can be made by the process disclosed in U.S. Pat. No.6,562,895. This isoprene-butadiene rubber is a liquid at roomtemperature and is comprised of repeat units which are derived fromabout 5 weight percent to about 95 weight percent isoprene and fromabout 5 weight percent to about 95 weight percent 1,3-butadiene, whereinthe repeat units derived from isoprene and 1,3-butadiene are inessentially random order. This IBR also has a low number averagemolecular weight which is within the range of about 3,000 to about50,000 and has a glass transition temperature which is within the rangeof about −50° C. to about 20° C.

These isoprene-butadiene copolymers are synthesized utilizing anorganolithium initiator and a polar modifier. The level of organolithiuminitiator employed will be dependent upon the molecular weight which isdesired for the liquid isoprene-butadiene polymer being synthesized. Asa general rule, in all anionic polymerizations the molecular weight ofthe polymer produced is inversely proportional to the amount ofinitiator utilized. Since liquid isoprene-butadiene polymer having arelatively low molecular weight is being synthesized, the amount ofinitiator employed will be relatively large. As a general rule, fromabout 0.1 to about 2 phm (parts per hundred parts of monomer by weight)of the organolithium compound will be employed. In most cases, it willbe preferred to utilize from about 0.2 to about 1 phm of theorganolithium compound with it being most preferred to utilize fromabout 0.4 phm to 0.6 phm of the organolithium compound. In any case, anamount of organolithium initiator will be selected to result in theproduction of liquid isoprene-butadiene polymer having a number averagemolecular weight which is within the range of about 3,000 to about50,000.

The amount of organolithium initiator will preferably be selected toresult in the production of liquid isoprene-butadiene polymer having anumber average molecular weight which is within the range of about 5,000to about 30,000. The amount of organolithium initiator will mostpreferably be selected to result in the production of liquidisoprene-butadiene polymer having a number average molecular weight thatis within the range of about 8,000 to about 18,000. In any case, it iscritical to carry out the copolymerization of the 1,3-butadiene and thestyrene in the presence of a polar modifier, such asN,N,N′,N′-tetramethylethylenediamine (TMEDA), to attain a high glasstransition temperature which is within the range of about −50° C. to 20°C. The teachings of U.S. Pat. No. 6,562,895 are incorporated herein byreference for the purpose illustrating the synthesis of liquidisoprene-butadiene polymers.

Block copolymers containing a block of polyisoprene can be made by theprocess described in U.S. Pat. No. 5,242,984. For instance, lineardiblock polymers of styrene and isoprene (S-I block copolymers) andlinear triblock polymers of styrene and isoprene (S-I-S triblockpolymers) can be made by this process. In this technique, the monomersare polymerized sequentially by anionic polymerization in an inertorganic solvent. Normally an organoalkali metal compound, such as analkyl lithium compound, is used to initiate the polymerization which canbe conducted over a broad temperature range.

Methods of controlling the molecular weights of the blocks and theoverall polymer are described in U.S. Pat. No. 3,149,182 and U.S. Pat.No. 3,231,635 which state that the amount of monomer can be keptconstant and different molecular weights can be achieved by changing theamount of catalyst or that the amount of catalyst can be kept constantand different molecular weights can be achieved by varying the amount ofthe monomer. Following the sequential polymerization, the product isterminated such as by the addition of a protic terminating agent, e.g.water, alcohol or other reagents or with hydrogen, for the purpose ofremoving the lithium radical forming the nucleus for the condensedpolymer product. The block polymer product is then recovered such as bycoagulation utilizing hot water or steam or both. The teachings of U.S.Pat. No. 5,242,984, U.S. Pat. No. 3,149,182, and U.S. Pat. No. 3,231,635are incorporated herein by reference for the purpose of teaching methodsfor synthesizing S-I block copolymers and S-I-S triblock polymers.

Carbon Fingerprinting

All types of polymers made with the isoprene of this invention areverifiable as being made with isoprene that did not originate from apetrochemical source. Additionally, the isoprene containing polymers ofthis invention can also be distinguished from isoprene containingpolymers that come from natural sources, such as natural rubber.Accordingly, the isoprene containing polymers of this invention areanalytically verifiable as coming from the bio-renewable,environmentally friendly, sources delineated herein.

Polymers derived from bioisoprene can be distinguished from polymersderived form petrochemical carbon on the basis of dual carbon-isotopicfingerprinting. Additionally, the specific source of biosourced carbon(e.g. glucose vs. glycerol) can be determined by dual carbon-isotopicfingerprinting (see, U.S. Pat. No. 7,169,588, which is hereinincorporated by reference).

This method usefully distinguishes chemically-identical materials, andapportions carbon in products by source (and possibly year) of growth ofthe biospheric (plant) component. The isotopes, ¹⁴C and ¹³C, bringcomplementary information to this problem. The radiocarbon datingisotope (¹⁴C), with its nuclear half life of 5730 years, clearly allowsone to apportion specimen carbon between fossil (“dead”) and biospheric(“alive”) feedstocks [Currie, L. A. “Source Apportionment of AtmosphericParticles,” Characterization of Environmental Particles, J. Buffle andH. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC EnvironmentalAnalytical Chemistry Series (Lewis Publishers, Inc) (1992) 3 74]. Thebasic assumption in radiocarbon dating is that the constancy of ¹⁴Cconcentration in the atmosphere leads to the constancy of ¹⁴C in livingorganisms. When dealing with an isolated sample, the age of a sample canbe deduced approximately by the relationship:t=(−5730/0.693)ln(A/A_(O)), where t=age, 5730 years is the half-life ofradiocarbon, and A and A_(O) are the specific ¹⁴C activity of the sampleand of the modem standard, respectively [Hsieh, Y., Soil Sci. Soc. AmJ., 56, 460, (1992)]. However, because of atmospheric nuclear testingsince 1950 and the burning of fossil fuel since 1850, ¹⁴C has acquired asecond, geochemical time characteristic. Its concentration inatmospheric CO₂—and hence in the living biosphere—approximately doubledat the peak of nuclear testing, in the mid-1960s. It has since beengradually returning to the steady-state cosmogenic (atmospheric)baseline isotope rate (¹⁴C/¹²C) of ca. 1.2×10⁻¹², with an approximaterelaxation “half-life” of 7-10 years. (This latter half-life must not betaken literally; rather, one must use the detailed atmospheric nuclearinput/decay function to trace the variation of atmospheric andbiospheric ¹⁴C since the onset of the nuclear age.) It is this latterbiospheric ¹⁴C time characteristic that holds out the promise of annualdating of recent biospheric carbon. ¹⁴C can be measured by acceleratormass spectrometry (AMS), with results given in units of “fraction ofmodem carbon” (f_(M)). f_(M) is defined by National Institute ofStandards and Technology (NIST) Standard Reference Materials (SRMs)4990B and 4990C, known as oxalic acids standards HOxI and HOxII,respectively. The fundamental definition relates to 0.95 times the¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughlyequivalent to decay-corrected pre-Industrial Revolution wood. For thecurrent living biosphere (plant material), f_(M)≈1.1.

The stable carbon isotope ratio (¹³C/¹²C) provides a complementary routeto source discrimination and apportionment. The ratio of carbon isotopes¹³C and ¹²C can be used to identify or rule out potential origins formany carbon-containing samples. This method works well because: (1) bothisotopes are stable on geological time frames; (2) the ratio of ¹³C to¹²C can be measured with great precision using combinations ofcombustion analysis, gas chromatography, and isotope ratio massspectrometry; (3) ¹³C/¹²C ratios for many naturally occurring materialsoccur within narrow ranges characteristic of those materials; and (4)¹³C/¹²C ratios for many materials change in predictable ways as thesematerials undergo chemical reactions.

Studies involving ¹³C/¹²C ratios at or near natural abundance levelsusually report isotopic data as “delta values”, which are represented bythe symbol δ¹³C and given in parts per thousand (‰) relative to astandard reference sample. For carbon, the reference sample typically isPee Dee Belemite, which has a ¹³C natural abundance of 1.112328% and isassigned δ¹³C 0.00‰. The formula relating ¹³C/¹²C ratios to delta valuesis:δ¹³C(in %)versus standard=[(R _(sample) −R _(standard))/R_(standard)](1000),where R_(sample) is the ¹³C/¹²C ratio for the sample and R_(standard) isthe ratio for Pee Dee Belemite.

Although isotopes of carbon (i.e., ¹³C and ¹²C) take part in the samephysical processes and same chemical reactions, the slight massdifference between ¹³C and ¹²C can be manifested in very slightdifferences in rates for many reactions and processes. This leads tosmall differences between ¹³C/¹²C ratios for samples subjected tochemical reactions or physical processes. For example, physicalprocesses such as evaporation or diffusion discriminate against heavierisotopes and typically lead to slight enrichment of the heavier isotopein the original sample as the lighter isotope evaporates or diffusesaway more rapidly. The ¹³C/¹²C ratio therefore increases slightly asevaporation or diffusion occurs. For chemical reactions, includingenzymatic reactions, the situation is more complex, but there often is aslight discrimination of one isotope over another, which can be detectedby measuring ¹³C/¹²C ratios or δ¹³C values. For example, atmospheric CO₂can be converted into plant matter via two very different mechanisms forphotosynthesis: the Calvin-Benson pathway, which occurs in C₃ plants,and the Hatch-Slack pathway, which occurs in C₄ plants. These twomechanisms are sufficiently different to produce a measurable differencein δ¹³C from the same CO₂. For C₄ plants, δ¹³C typically ranges from −9‰to −17‰ with a mean near −13‰. For C₃ plants, δ¹³C typically ranges from−20‰ to −32‰ with a mean near −27‰. Because these ranges are sodifferent and δ¹³C values can be routinely measured within 0.02‰, it isrelatively easy to distinguish between plant residues derived from C₃versus C₄ plants. This has myriad applications in archeology and otherfields where analysis of carbon-containing residues from cooking orskeletal remains can be used to track the evolution, activities anddiets of humans and other animals.

More recently, δ¹³C values have been utilized to detect economic fraud,especially the adulteration of foodstuffs by other materials—includingpotentially harmful synthetics derived from petrochemicals. For example,maize (corn) oil is considered to be a premium vegetable oil and thereis a temptation for unscrupulous producers to dilute maize oil withcheaper oils. Fortunately, maize oil is derived from a C₄ plant whilemost of the cheaper alternatives are derived from C₃ plants or animals.The δ¹³C for authentic maize oil is therefore −13.7‰ to −16.4‰ comparedto −25‰ to −32‰ for the alternatives. Any significant dilution of maizeoil by a cheaper alternative can be detected by measuring δ¹³C.Similarly, the addition of cane sugar (a product of C₄ photosynthesis)to fruit juices, wines, spirits, and honey (all products of C₃photosynthesis) can be detected by measuring δ¹³C values. It is evenpossible to detect the adulteration of natural flavors by syntheticanalogs and the use of illegal synthetic hormone supplements via δ¹³Cvalues.

The current invention utilizes the ability to accurately measure δ¹³Cvalues in order to produce new, isotopically unique isoprenic polymersthat can be readily distinguished from polymers derived frompetroleum-based feedstocks. The current invention also utilizes theability to accurately measure δ¹³C values in order to produce new,isotopically unique isoprenic polymers that can be readily distinguishedfrom natural rubber. A salient feature of the current invention is thatit provides new polymers with a broad range of δ¹³C values that can betailored and subsequently verified for authenticity. As describedearlier, these new polymers satisfy an increasing need from customersfor verifiable products that contain neither potential proteinaceousallergens nor feedstocks derived from petroleum.

The polymers represented by the current invention contain isoprene unitsthat are isotopically unique compared to both natural rubber andsynthetic polymers containing petroleum-derived isoprene. In the case ofnatural rubber derived from Hevea brasiliensis (i.e., the common naturalrubber tree), δ¹³C values typically range from about −27‰ to about −28‰.Guayule rubber, which is derived from a desert shrub, has δ¹³C of about−31‰. Both rubbers exhibit δ¹³C values expected for products of C₃photosynthesis, and both rubbers are known to contain polymer-boundproteins.

Traditional synthetic polyisoprene can have different δ¹³C valuesdepending on the source of isoprene. For isoprene derived fromextractive distillation of C₅ streams from petroleum refineries, δ¹³C isabout −22‰ to about −24‰. This range is typical for light, unsaturatedhydrocarbons derived from petroleum, and polymers containingpetroleum-based isoprene typically contain isoprenic units with the sameδ¹³C. For polymers containing isoprene derived from the reaction ofisobutylene with formaldehyde, δ¹³C values can be about −34.4‰ becauseformaldehyde is often derived from feedstocks with much more negativeδ¹³C values.

The current invention provides isoprene-containing polymers with verydifferent δ¹³C values. For example, fermentation of corn-derived glucose(δ¹³C −10.73‰) with minimal amounts of other carbon-containing nutrients(e.g., yeast extract) produces isoprene which can be polymerized intopolyisoprene with δ¹³C −14.66‰ to −14.85‰. The δ¹³C for this polymerclearly is in a new range that is well outside the normal ranges fornatural rubber and all previously known synthetic polyisoprene, and itis within the range normally associated with products derived from C₄plants. The unique δ¹³C value for this polymer is a direct consequenceof the fact that the isoprene in the polymer is derived from corn-basedglucose, which indeed is a product derived from C₄ plants.

It is recognized by those with ordinary skill in the art that similarresults can be obtained using other sugars or fermentable derived fromC₄ plants. For example, sucrose from sugar cane (δ¹³C −10.4%‰, invertsugar from sugar cane (δ¹³C −15.3%‰, glucose from cornstarch (δ¹³C−11.1‰), and glucose from hydrolytic degradation of either corn stover(δ¹³C −11.3‰) or sugar cane bagasse (δ¹³C −13.0‰ should all produceisoprene that can be used to produce isoprene polymers with δ¹³C valuesthat are less negative than either natural rubber or synthetic polymerscontaining petroleum-based isoprene. Those with ordinary skill in theart also will recognize that it should be possible to produce isopreneand isoprene polymers with δ¹³C less negative than about −22‰ fromfermentable feedstocks with δ¹³C approximately greater (i.e., lessnegative) than about −18‰, including mixtures of fermentable feedstockswith an average δ¹³C approximately greater than about −18‰.

In addition to producing isoprene-containing polymers with δ¹³C valuescharacteristic of products derived from C₄ plants, those skilled in theart will recognize that uniquely isotopically labeledisoprene-containing polymers can be made from fermentable non-C₄feedstocks. For example, glucose from hydrolyzed softwood pulp (δ¹³C−23‰ should yield isoprene and polyisoprene with δ¹³C near −27‰, whichis in a unique range between the normal ranges observed for isoprenederived from extractive distillation of C₅ fractions and isoprenederived from the reaction of isobutylene with formaldehyde. Thoseskilled in the art also will recognize that fermentation of other sugarswith δ¹³C ranges of approximately −20‰ to about −28‰ should produceisoprene and isoprenic polymers with δ¹³C ranging from about −24‰ toabout −32‰. These other sugars might include (but are not limited to)glucose from hydrolyzed cellulose (δ¹³C −25±2%‰, invert sugar from beetsugar (δ¹³C −26‰ to −27‰), and lactose (δ¹³C −27‰ to −28‰). Fermentationof plant oils (δ¹³C −26‰ to −32‰, including palm oil (δ¹³C −30‰ couldprovide access to isoprene polymers with δ¹³C more negative than −30‰.

Those skilled in the art will recognize that cofermentation of two ormore feedstocks can be used to produce isoprene and thereforeisoprene-containing polymers with intermediate δ¹³C values. For example,a 1:1 mixture of sucrose from sugar cane (δ¹³C −10.4‰ and sucrose frombeet sugar (δ¹³C −26‰ to −27‰) should produce isoprene and thereforeisoprene-containing polymers with approximately the same δ¹³C value aspolymer produced from sucrose derived from a single source with theaverage δ¹³C value (i.e., approx −18.5‰). The same should be true forinvert sugars derived from sugar and beets. In both cases, it should beobvious that the same polymers could be synthesized by mixing and then(co)polymerizing equal amounts of isoprene separately prepared fromsucrose or invert sugar derived from sugar cane and beets. It alsoshould be obvious that cofermentation of sugars with other fermentablefeedstocks—such as yeast extract and plant oils—can be used to produceisoprene and therefore isoprene-containing polymers with intermediateδ¹³C values. For example, cofermentation of glucose (δ¹³C −10.73‰) andyeast extract (δ¹³C −26‰ to −27‰) in a ratio of 181.2:17.6 producesisoprene which can be polymerized to polyisoprene with δ¹³C values of−18‰ to −20‰. In contrast, fermentation of glucose with a minimal amountof yeast extract and subsequent polymerization of the isoprene producespolyisoprene with δ¹³C values of −14‰ to −15‰.

For copolymers of isoprene with other monomers, those skilled in the artrecognize that there is a finite amount of isoprene that is incorporatedinto the polymer background as “blocks” of polyisoprene. The tendency ofisoprene to form blocks of two or more isoprenic units—even in “randomcopolymers”—depends on many factors, including the amount of isoprenerelative to other monomers, the type of catalyst used forpolymerization, and the specific reaction conditions for polymerization.The presence of these blocks along the polymer backbone can usually bedetected by NMR spectroscopy. By using a combination of chemicaldegradation (e.g., ozonolysis) and chromatography, it is possible toisolate fragments of these blocks for chemical analysis, includingmeasurement of δ¹³C values for the blocks derived from isoprene. Thisprovides a way for determining whether copolymers of isoprene with othermonomers contain isoprene derived from renewable/sustainable feedstocks,especially feedstocks derived from C₄ plants.

The polyisoprene polymers of this invention which are made with isoprenemonomer from the cells cultures that utilize bio-renewable carbonsources can be identified as such by virtue of their δ¹³C value andother polymer characteristics. For instance, the following isoprenecontaining polymers are verifiable as containing isoprene monomer thatwas produced utilizing the method of this invention:

(1) Polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has δ¹³Cvalue of greater than −22‰. Such polyisoprene polymers can have a δ¹³Cvalue which is greater than −21‰, and can also have a δ¹³C value whichis greater than −20‰. In some cases, the polyisoprene polymer will has aδ¹³C value which is within the range of −22‰ to −10‰, and in other casesit will have a δ¹³C value which is within the range of −21‰ to −12‰. Instill other cases the polyisoprene polymer will have a δ¹³C value whichis within the range of −20‰ to −14‰. In many cases, the polyisoprenepolymer will be polyisoprene homopolymer rubber.

(2) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has δ¹³Cvalue which is within the range of −30‰ to −28.5‰. Such polyisoprenepolymers can have a δ¹³C value which is within the range of −30‰ to−29‰. In some cases, the polyisoprene polymer will have a δ¹³C valuewhich is within the range of −30‰ to −29‰, and in other cases thepolyisoprene polymer will have a δ¹³C value which is within the range of−30‰ to −29.5‰. In still other cases the polyisoprene polymer can have aδ¹³C value which is within the range of −29.5‰ to −28.5‰ and in stillfurther cases the polyisoprene polymer can have a δ¹³C value which iswithin the range of −29.0‰ to −28.5‰. In many cases, the polyisoprenepolymer will be polyisoprene homopolymer rubber.

(3) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene is free ofprotein, and wherein the polyisoprene polymer has δ¹³C value which iswithin the range of −34‰ to −24‰. In some cases this polyisoprenepolymer has δ¹³C value which is within the range of −32‰ to −25‰. Insome cases this polyisoprene polymer has δ¹³C value which is within therange of −34‰ to −25‰. In other cases the polyisoprene polymer has aδ¹³C value which is within the range of −33‰ to −25‰, and in still othercases the polyisoprene polymer has a δ¹³C value which is within therange of −32‰ to −25‰. In many cases, the polyisoprene polymer will bepolyisoprene homopolymer rubber.

(4) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has acis-1,4-microstructure content of less than 99.9%, wherein thepolyisoprene polymer has a trans-1,4-microstructure content of less than99.9%, and wherein the polyisoprene polymer has δ¹³C value of which iswithin the range of −34‰ to −24‰. Such polyisoprene can have a δ¹³Cvalue which is within the range of −34‰ to −25‰. In some cases thepolyisoprene polymer will have a δ¹³C value which is within the range of−33‰ to −25‰. In other cases the polyisoprene polymer will have a δ¹³Cvalue which is within the range of −32‰ to −25‰. In other cases thepolyisoprene polymer will have a δ¹³C value which is within the range of−32‰ to −24‰. The polyisoprene polymer can have a cis-1,4-microstructurecontent of less than 99.8%. In other cases the polyisoprene polymer willhave a cis-1,4-microstructure content of less than 99.7%. In still othercases the polyisoprene polymer will have a cis-1,4-microstructurecontent of less than 99.5% or even less than 99%. In many cases thepolyisoprene polymer will have a cis-1,4-microstructure content of lessthan 98.5% or even less than 98%. This polyisoprene polymer can alsohave a polydispersity of less than 2.0 or even less than 1.8. In somecases the polyisoprene polymer has a polydispersity of less than 1.6 oreven less than 1.5. In still other cases the polyisoprene polymer canhave a polydispersity of less than 1.4 or even less than 1.2. In manycases the polyisoprene polymer will have a polydispersity of less than1.1.

(5) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has a3,4-microstructure content of greater than 2%, and wherein thepolyisoprene polymer has δ¹³C value of which is within the range of −34‰to −24‰. Such polyisoprene polymers can have a δ¹³C value which iswithin the range of −34‰ to −25‰. In some cases the polyisoprene polymerwill have a δ¹³C value which is within the range of −33‰ to −25‰. Inother cases polyisoprene polymer will have a δ¹³C value which is withinthe range of −32‰ to −25‰. In other cases polyisoprene polymer will havea δ¹³C value which is within the range of −32‰ to −24‰. The polyisoprenepolymer can have a 3,4-microstructure content of greater than 5%. Insome cases the polyisoprene polymer will have a 3,4-microstructurecontent of greater than 10%. In other cases the polyisoprene polymerwill have a 3,4-microstructure content of greater than 15%. In stillother the polyisoprene polymer will have a 3,4-microstructure content ofgreater than 20%. In many cases the polyisoprene polymer will have a3,4-microstructure content of greater than 25%. This polyisoprenepolymer can have a polydispersity of less than 2.0. In some cases thepolyisoprene polymer will have a polydispersity of less than 1.8. Inother cases the polyisoprene polymer will have a polydispersity of lessthan 1.6. In still other cases the polyisoprene polymer will have apolydispersity of less than 1.5 or even than 1.4. In many cases thepolyisoprene polymer will have a polydispersity of less than 1.2 or evenless than 1.1.

(6) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has a1,2-microstructure content of greater than 2%, and wherein thepolyisoprene polymer has δ¹³C value of which is within the range of −34‰to −24‰. Polyisoprene polymers of this type can have a δ¹³C value whichis within the range of −34‰ to −25‰. In some cases, the polyisoprenepolymer will have a δ¹³C value which is within the range of −33‰ to−25‰. In other cases, the polyisoprene polymer will have a δ¹³C valuewhich is within the range of −32‰ to −25‰. In other cases, thepolyisoprene polymer will have a δ¹³C value which is within the range of−32‰ to −24‰. The polyisoprene polymer can have a 1,2-microstructurecontent of greater then than 5%. In some cases, the polyisoprene polymerwill have a 1,2-microstructure content of greater than 10%. In othercases, the polyisoprene polymer will have a 1,2-microstructure contentof greater than 15%. In still other cases, the polyisoprene polymer willhave a 1,2-microstructure content of greater than 20%. In many cases,the polyisoprene polymer will have a 1,2-microstructure content ofgreater than 25%. The polyisoprene polymer can have a polydispersity ofless than 2.0. In some cases, the polyisoprene polymer will have apolydispersity of less than 1.8. In other cases, the polyisoprenepolymer will have a polydispersity of less than 1.6. In still othercases, the polyisoprene polymer will have a polydispersity of less than1.5. In many cases, the polyisoprene polymer will have a polydispersityof less than 1.4 or even less than 1.2. It is possible for thepolyisoprene polymer to have a polydispersity of less than 1.1.

(7) A polymer which is comprised of repeat units that are derived fromisoprene monomer and at least one additional monomer, wherein thepolymer includes blocks of repeat units that are derived from isoprene,and wherein the blocks of repeat units that are derived from isoprenehave a δ¹³C value of greater than −22‰. Such polyisoprene polymers canhave a δ¹³C value which is greater than −21‰. In some cases, thepolyisoprene polymer will have a δ¹³C value which is greater than −20‰.In other cases, the polyisoprene polymer will have a δ¹³C value which iswithin the range of −22‰ to −10‰. In still other cases, the polyisoprenepolymer will have a δ¹³C value which is within the range of −21‰ to−12‰. In many cases, the polyisoprene polymer will have a δ¹³C valuethat is within the range of −20‰ to −14‰.

(8) A polymer which is comprised of repeat units that are derived fromisoprene monomer and at least one additional monomer, wherein thepolymer includes blocks of repeat units that are derived from isoprene,and wherein the blocks of repeat units that are derived from isoprenehave a δ¹³C value which is within the range of −34‰ to −24‰. Suchcopolymers can have a δ¹³C value is within the range of −34‰ to −25‰. Insome cases, copolymer of this type will have a δ¹³C value which iswithin the range of −33‰ to −25‰. In other cases, copolymers of thistype will have a δ¹³C value is within the range of −32‰ to −25‰. Inother cases, copolymers of this type will have a δ¹³C value is withinthe range of −32‰ to −24‰. Copolymers of this type can be rubberycopolymers of isoprene and 1,3-butadiene, rubbery copolymer of isopreneand styrene, rubbery copolymers of isoprene and α-methyl styrene, andthe like.

(9) A liquid polyisoprene polymer which is comprised of repeat unitsthat are derived from isoprene monomer, wherein the polyisoprene polymerhas weight average molecular weight which is within the range of 5,000to 100,000, and wherein the liquid polyisoprene polymer has δ¹³C valueof which is within the range of −34‰ to −24‰. Such liquid polyisoprenepolymers can have a δ¹³C value which is within the range of −34‰ to−25‰. In some cases, the liquid polyisoprene polymer will have a δ¹³Cvalue which is within the range of −33‰ to −25‰. In other cases, theliquid polyisoprene polymer will have a δ¹³C value which is within therange of −32‰ to −25‰. In other cases, the liquid polyisoprene polymerwill have a δ¹³C value which is within the range of −32‰ to −24‰. Suchliquid polyisoprene polymers can have a weight average molecular weightthat is within the range of 20,000 to 80,000. In some cases, the liquidpolyisoprene polymer will have a weight average molecular weight whichis within the range of 30,000 to 50,000. In other cases, thepolyisoprene polymer will have a polydispersity of less than 2.0 or evenless than 1.8. In still other cases, the liquid polyisoprene polymerwill have a polydispersity of less than 1.6 or even less than 1.5. Inmany cases, the liquid polyisoprene polymer will have a polydispersityof less than 1.4 or even less than 1.2. It is possible for the liquidpolyisoprene polymer to have a polydispersity of less than 1.1.

(10) A liquid polyisoprene polymer which is comprised of repeat unitsthat are derived from isoprene monomer, wherein the liquid polyisoprenepolymer has a weight average molecular weight which is within the rangeof 5,000 to 100,000, and wherein the liquid polyisoprene polymer hasδ¹³C value of which is within the range of −34‰ to −24‰. Such liquidpolyisoprene polymers can have a δ¹³C value which is within the range of−34‰ to −25‰. In some cases, the liquid polyisoprene polymer will have aδ¹³C value which is within the range of −33‰ to −25‰. In still othercases, the liquid polyisoprene polymer will have a δ¹³C value which iswithin the range of −32‰ to −25‰. In other cases, the liquidpolyisoprene polymer will have a δ¹³C value which is within the range of−32‰ to −24‰. Such liquid polyisoprene can have a weight averagemolecular weight that is within the range of 20,000 to 80,000. Theliquid polyisoprene will typically have a weight average molecularweight which is within the range of 30,000 to 50,000. Such liquidpolyisoprene can have a polydispersity of less than 2.0. In some cases,the liquid polyisoprene polymer will have a polydispersity of less than1.8. In other cases, the liquid polyisoprene polymer has apolydispersity of less than 1.6. In still other cases, the liquidpolyisoprene polymer will have a polydispersity of less than 1.5 or evenless than 1.4. In many cases, the liquid polyisoprene polymer will havea polydispersity of less than 1.2 or even less than 1.1.

The polyisoprene homopolymer, liquid polyisoprene polymer orpolyisoprene co-polymer, or any variations described herein, produced bychemical polymerization of isoprene derived from renewable resources canbe distinguished from products derived from petrochemical resources byits ¹⁴C content. In some embodiments, a polymer derived from bioisoprenecomprises radioactive carbon-14. In some embodiments, the ¹⁴C/¹²C ratiois greater than or about 1.0×10⁻¹², 1.05×10⁻¹², 1.1×10⁻¹², 1.15×10⁻¹²,or 1.2×10⁻¹². In some embodiments, the polymer derived from bioisoprenehas an f_(M) value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1.In some embodiments, the polymer derived from bioisoprene has an f_(M)value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1 and δ¹³Cvalues of greater (less negative) than −22‰. In some embodiments, thepolymer derived from bioisoprene has an f_(M) value of greater than orabout 0.9, 0.95, 1.0, 1.05 or 1.1 and a δ¹³C value which is within therange of −22 to −10, −21 to −12, or −20 to −14‰. In some embodiments,the polymer derived from bioisoprene has an f_(M) value of greater thanor about 0.9, 0.95, 1.0, 1.05 or 1.1 and a δ¹³C value which is withinthe range of −34 to −24, −32 to −24, −34 to −25, −33 to −25, −32 to −25,−30 to −29, −30.0 to −29.5, −29.5 to −28.5, or −29.0 to −28.5‰.

This invention is illustrated by the following examples that are merelyfor the purpose of illustration and are not to be regarded as limitingthe scope of the invention or the manner in which it can be practiced.Unless specifically indicated otherwise, parts and percentages are givenby weight.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. Unless indicated otherwise, temperature is in degreesCentigrade and pressure is at or near atmospheric pressure. Theforegoing examples and detailed description are offered by way ofillustration and not by way of limitation. All publications, patentapplications, and patents cited in this specification are hereinincorporated by reference as if each individual publication, patentapplication, or patent were specifically and individually indicated tobe incorporated by reference. In particular, all publications citedherein are expressly incorporated herein by reference for the purpose ofdescribing and disclosing compositions and methodologies which might beused in connection with the invention. Although the foregoing inventionhas been described in some detail by way of illustration and example forpurposes of clarity of understanding, it will be readily apparent tothose of ordinary skill in the art in light of the teachings of thisinvention that certain changes and modifications may be made theretowithout departing from the spirit or scope of the appended claims.

In the practice of this invention ¹³C analysis can be done by loading0.5 to 1.0 mg samples into tin cups for carbon isotopic analysis using aCostech ECS4010 Elemental Analyzer as an inlet for a ThermoFinniganDelta Plus XP isotope ratio mass spectrometer. Samples are dropped intoa cobaltous/cobaltic oxide combustion reactor at 1020° C. withcombustion gases being passed in a helium stream at 85 mL/min through acopper reactor (650° C.) to convert NO_(x) to N₂. CO₂ and N₂ areseparated using a 3-m 5 Å molecular sieve column. Then, ¹³C/¹²C ratiosare calibrated to the VPDB scale using two laboratory standards(Acetanilide B, −29.52±0.02‰ m and cornstarch A, −11.01±0.02‰) whichhave been carefully calibrated to the VPDB scale by off-line combustionand dual-inlet analysis using the 2-standard approach of T. B. Coplen etal, New Guidelines for δ¹³C Measurements, Anal. Chem., 78, 2439-2441(2006). The teachings of Coplen are incorporated herein by reference forthe purpose of teaching the technique for determining δ¹³C values.

Example 1 Production of Isoprene in E. Coli Expressing Recombinant KudzuIsoprene Synthase

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein E. Coli

The protein sequence for the kudzu (Pueraria montana) isoprene synthasegene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprenesynthase gene, optimized for E. coli codon usage, was purchased fromDNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from thesupplied plasmid by restriction endonuclease digestion withBspLU11I/PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen)that had been digested with NcoI/PstI. The construct was designed suchthat the stop codon in the isoprene synthase gene 5′ to the PstI site.As a result, when the construct was expressed the His-Tag is notattached to the isoprene synthase protein. The resulting plasmid,pTrcKudzu, was verified by sequencing (FIGS. 2 and 3).

The isoprene synthase gene was also cloned into pET16b (Novagen). Inthis case, the isoprene synthase gene was inserted into pET16b such thatthe recombinant isoprene synthase protein contained the N-terminal Histag. The isoprene synthase gene was amplified from pTrcKudzu by PCRusing the primer set pET-His-Kudzu-2F:5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:3) andpET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:4). These primers added an NdeI site at the 5′-end and a BamH1 siteat the 3′ end of the gene respectively. The plasmid pTrcKudzu, describedabove, was used as template DNA, Herculase polymerase (Stratagene) wasused according to manufacture's directions, and primers were added at aconcentration of 10 pMols. The PCR was carried out in a total volume of25 μl. The PCR product was digested with NdeI/BamH1 and cloned intopET16b digested with the same enzymes. The ligation mix was transformedinto E. coli Top10 (Invitrogen) and the correct clone selected bysequencing. The resulting plasmid, in which the kudzu isoprene synthasegene was expressed from the T7 promoter, was designated pETNHisKudzu(FIGS. 4 and 5).

The kudzu isoprene synthase gene was also cloned into the low copynumber plasmid pCL1920. Primers were used to amplify the kudzu isoprenesynthase gene from pTrcKudzu described above. The forward primer added aHindIII site and an E. coli consensus RBS to the 5′ end. The PstIcloning site was already present in pTrcKudzu just 3′ of the stop codonso the reverse primer was constructed such that the final PCR productincludes the PstI site. The sequences of the primers were:HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQID NO:6) and BamH1-Kudzu R:

5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). The PCR productwas amplified using Herculase polymerase with primers at a concentrationof 10 pmol and with 1 ng of template DNA (pTrcKudzu). The amplificationprotocol included 30 cycles of (95° C. for 1 minute, 60° C. for 1minute, 72° C. for 2 minutes). The product was digested with HindIII andPstI and ligated into pCL1920 which had also been digested with HindIIIand PstI. The ligation mix was transformed into E. coli Top10. Severaltransformants were checked by sequencing. The resulting plasmid wasdesignated pCL-lac-Kudzu (FIGS. 6 and 7).

II. Determination of Isoprene Production

For the shake flask cultures, one ml of a culture was transferred fromshake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753;cap cat#5188 2759). The cap was screwed on tightly and the vialsincubated at the equivalent temperature with shaking at 250 rpm. After30 minutes the vials were removed from the incubator and analyzed asdescribed below (see Table 1 for some experimental values from thisassay).

In cases where isoprene production in fermentors was determined, sampleswere taken from the off-gas of the fermentor and analyzed directly asdescribed below (see Table 2 for some experimental values from thisassay).

The analysis was performed using an Agilent 6890 GC/MS system interfacedwith a CTC Analytics (Switzerland) CombiPAL autosampler operating inheadspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μmfilm thickness) was used for separation of analytes. The sampler was setup to inject 500 μL of headspace gas. The GC/MS method utilized heliumas the carrier gas at a flow of 1 ml/minutes. The injection port washeld at 250° C. with a split ratio of 50:1. The oven temperature washeld at 37° C. for the 2 minute duration of the analysis. The Agilent5793N mass selective detector was run in single ion monitoring (SIM)mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes toallow the elution of permanent gases. Under these conditions isoprene(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. Acalibration table was used to quantify the absolute amount of isopreneand was found to be linear from 1 μg/L to 200 μg/L. The limit ofdetection was estimated to be 50 to 100 ng/L using this method.

III. Production of Isoprene in Shake Flasks Containing E. coli CellsExpressing Recombinant Isoprene Synthase

The vectors described above were introduced to E. coli strain BL21(Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu andBL21/pETHisKudzu. The strains were spread for isolation onto LA (Luriaagar) and carbenicillin (50 μg/ml) and incubated overnight at 37° C.Single colonies were inoculated into 250 ml baffled shake flaskscontaining 20 ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml).Cultures were grown overnight at 20° C. with shaking at 200 rpm. TheOD₆₀₀ of the overnight cultures were measured and the cultures werediluted into a 250 ml baffled shake flask containing 30 ml MagicMedia(Invitrogen) and carbenicillin (100 μg/ml) to an OD₆₀₀˜0.05. The culturewas incubated at 30° C. with shaking at 200 rpm. When the OD₆₀₀˜0.5-0.8,400 μM IPTG was added and the cells were incubated for a further 6 hoursat 30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours afterinduction with IPTG, 1 ml aliquots of the cultures were collected, theOD₆₀₀ was determined and the amount of isoprene produced was measured asdescribed above. Results are shown in FIG. 8.

IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation

Large scale production of isoprene from E. coli containing therecombinant kudzu isoprene synthase gene was determined from a fed-batchculture. The recipe for the fermentation media (TM2) per liter offermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g,MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) andq.s. to volume. The final product was filter sterilized with 0.22μfilter (only, not autoclaved). The recipe for 1000× Modified Trace MetalSolution was as follows: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg,H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22μ filter.

This experiment was carried out in 14 L bioreactor to monitor isopreneformation from glucose at the desired fermentation, pH 6.7 andtemperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu takenfrom a frozen vial was prepared in soytone-yeast extract-glucose medium.After the inoculum grew to OD₅₅₀=0.6, two 600 ml flasks were centrifugedand the contents resuspended in 70 ml supernatant to transfer the cellpellet (70 ml of OD 3.1 material) to the bioreactor. At various timesafter inoculation, samples were removed and the amount of isopreneproduced was determined as described above. Results are shown in FIG. 9.

Example 2 Production of Isoprene in E. coli Expressing RecombinantPoplar Isoprene Synthase

The protein sequence for the poplar (Populus alba x Populus tremula)isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786)was obtained from GenBank (CAC35696). A gene, codon optimized for E.coli, was purchased from DNA2.0 (p9796-poplar, FIGS. 30 and 31). Theisoprene synthase gene was removed from the supplied plasmid byrestriction endonuclease digestion with BspLU11I/PstI, gel-purified, andligated into pTrcHis2B that had been digested with NcoI/PstI. Theconstruct is cloned such that the stop codon in the insert is before thePstI site, which results in a construct in which the His-Tag is notattached to the isoprene synthase protein. The resulting plasmidpTrcPoplar (FIGS. 32 and 33), was verified by sequencing.

Example 3 Production of Isoprene in Panteoa citrea ExpressingRecombinant Kudzu Isoprene Synthase

The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 wereelectroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformantswere selected on LA containing carbenicillin (200 μg/ml) orspectinomycin (50 μg/ml) respectively. Production of isoprene from shakeflasks and determination of the amount of isoprene produced wasperformed as described in Example 1 for E. coli strains expressingrecombinant kudzu isoprene synthase. Results are shown in FIG. 10.

Example 4 Production of Isoprene in Bacillus subtilis ExpressingRecombinant Kudzu Isoprene Synthase

I. Construction of a B. Subtilis Replicating Plasmid for the Expressionof Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was expressed in Bacillus subtilisaprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid(pBS19 with a chloramphenicol resistance cassette) under control of theaprE promoter. The isoprene synthase gene, the aprE promoter and thetranscription terminator were amplified separately and fused using PCR.The construct was then cloned into pBS19 and transformed into B.subtilis.

a) Amplification of the aprE Promoter

The aprE promoter was amplified from chromosomal DNA from Bacillussubtilis using the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 58)5′-GACATCAATTGCTCCATTTTCTTCTGCTATC CF 07-43 (−) Fuse aprE promoter toKudzu ispS (SEQ ID NO: 59) 5′-ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTA

b) Amplification of the Isoprene Synthase Gene

The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu(SEQ ID NO:2). The gene had been codon optimized for E. coli andsynthesized by DNA 2.0. The following primers were used:

CF 07-42 (+) Fuse the aprE promoter to kudzu isoprene synthase gene (GTGstart codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-45 (−) Fuse the3′ end of kudzu isoprene synthase gene to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC

c) Amplification of the Transcription Terminator

The terminator from the alkaline serine protease of Bacillusamyliquefaciens was amplified from a previously sequenced plasmidpJHPms382 using the following primers:

CF 07-44 (+) Fuse the 3′ end of kudzu isoprenesynthase to the terminator (SEQ ID NO: 62)5′-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGGCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu fragment was fused to the terminator fragment using PCR withthe following primers:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAATCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu-terminator fragment was fused to the promoter fragment usingPCR with the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 58)5′-GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes MfeI and BamHI. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated to a vector known aspBS19, which had been digested with EcoRI and BamHI and gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA and 50 carbenicillin plates. A total of six colonieswere chosen and grown overnight in LB and 50 carbenicillin and thenplasmids were isolated using a Qiagen kit. The plasmids were digestedwith EcoRI and BamHI to check for inserts and three of the correctplasmids were sent in for sequencing with the following primers:

CF 149 (+) EcoRI start of aprE promoter (SEQ ID NO: 65)5′-GACATGAATTCCTCCATTTTCTTCTGC CF 847 (+) Sequence in pXX 049(end of aprE promoter) (SEQ ID NO: 66) 5′-AGGAGAGGGTAAAGAGTGAGCF 07-45 (−) Fuse the 3′ end of kudzu isoprenesynthase to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATCCF 07-48 (+) Sequencing primer for kudzu isoprene synthase(SEQ ID NO: 67) 5′-CTTTTCCATCACCCACCTGAAGCF 07-49 (+) Sequencing in kudzu isoprene synthase (SEQ ID NO: 68)5′-GGCGAAATGGTCCAACAACAAAATTATC

The plasmid designated pBS Kudzu #2 (FIGS. 52 and 12) was correct bysequencing and was transformed into BG 3594 comK, a Bacillus subtilishost strain. Selection was done on LA and 5 chloramphenicol plates. Atransformant was chosen and struck to single colonies on LA and 5chloramphenicol, then grown in LB and 5 chloramphenicol until it reachedan OD₆₀₀ of 1.5. It was stored frozen in a vial at −80° C. in thepresence of glycerol. The resulting strain was designated CF 443.

II. Production of Isoprene in Shake Flasks Containing B. subtilis CellsExpressing Recombinant Isoprene Synthase.

Overnight cultures were inoculated with a single colony of CF 443 from aLA and Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB and Cmat 37° C. with shaking at 200 rpm. These overnight cultures (1 ml) wereused to inoculate 250 ml baffled shake flasks containing 25 ml Grants IImedia and chloramphenicol at a final concentration of 25 μg/ml. GrantsII Media recipe was 10 g soytone, 3 ml 1M K₂HPO₄, 75 g glucose, 3.6 gurea, 100 ml 10×MOPS, q.s. to 1 L with H₂O, pH 7.2; 10×MOPS recipe was83.72 g MOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K₂SO₄solution, 10 ml 0.528M MgCl₂ solution, 29.22 g NaCl, 100 ml 100×micronutrients, q.s. to 1 L with H₂O; and 100× micronutrients recipe was1.47 g CaCl₂*2H₂O, 0.4 g FeSO₄*7H₂0, 0.1 g MnSO₄*H₂0, 0.1 g ZnSO₄*H₂O,0.05 g CuCl₂*2H₂O, 0.1 g CoCl₂*6H₂O, 0.1 g Na₂MoO₄.2H₂O, q.s. to 1 Lwith H₂O, Shake flasks were incubated at 37° C. and samples were takenat 18, 24, and 44 hours. At 18 hours the headspaces of CF443 and thecontrol strain were sampled. This represented 18 hours of accumulationof isoprene. The amount of isoprene was determined by gas chromatographyas described in Example 1. Production of isoprene was enhancedsignificantly by expressing recombinant isoprene synthase (FIG. 11).

III. Production of Isoprene by CF443 in 14 L Fermentation

Large scale production of isoprene from B. subtilis containing therecombinant kudzu isoprene synthase gene on a replication plasmid wasdetermined from a fed-batch culture. Bacillus strain CF 443, expressinga kudzu isoprene synthase gene, or control stain which does not expressa kudzu isoprene synthase gene were cultivated by conventional fed-batchfermentation in a nutrient medium containing soy meal (Cargill), sodiumand potassium phosphate, magnesium sulfate and a solution of citricacid, ferric chloride and manganese chloride. Prior to fermentation themedia is macerated for 90 minutes using a mixture of enzymes includingcellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batchfermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADMVersadex greens or Danisco invert sugar) and 99% wt/wt oil (WesternFamily soy oil, where the 99% wt/wt is the concentration of oil beforeit was added to the cell culture medium). Feed was started when glucosein the batch was non-detectable. The feed rate was ramped over severalhours and was adjusted to add oil on an equal carbon basis. The pH wascontrolled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case offoaming, antifoam agent was added to the media. The fermentationtemperature was controlled at 37° C. and the fermentation culture wasagitated at 750 rpm. Various other parameters such as pH, DO %, airflow,and pressure were monitored throughout the entire process. The DO % ismaintained above 20. Samples were taken over the time course of 36 hoursand analyzed for cell growth (OD₅₅₀) and isoprene production. Results ofthese experiments are presented in FIGS. 53A and 53B.

IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis.

The kudzu isoprene synthase gene was cloned in an integrating plasmid(pJH101-cmpR) under the control of the aprE promoter. Under theconditions tested, no isoprene was detected.

Example 5 Production of Isoprene in Trichoderma

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Trichoderma reesei

The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized byDNA 2.0 (SEQ ID NO:8) (FIG. 13). This plasmid served as the template forthe following PCR amplification reaction: 1 μl plasmid template (20ng/ul), 1 μl Primer EL-945 (10 μM)5′-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:9), 1 μl PrimerEL-965 (10 μM) 5′-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:10), 1 μldNTP (10 mM), 5 μl 10×PfuUltra II Fusion HS DNA Polymerase Buffer, 1 μlPfuUltra II Fusion HS DNA Polymerase, 40 μl water in a total reactionvolume of 50 μl. The forward primer contained an additional 4nucleotides at the 5′-end that did not correspond to the Y. lipolyticacodon-optimized kudzu isoprene synthase gene, but was required forcloning into the pENTR/D-TOPO vector. The reverse primer contained anadditional 21 nucleotides at the 5′-end that did not correspond to theY. lipolytica codon-optimized kudzu isoprene synthase gene, but wereinserted for cloning into other vector backbones. Using the MJ ResearchPTC-200 Thermocycler, the PCR reaction was performed as follows: 95° C.for 2 minutes (first cycle only), 95° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 30 seconds (repeat for 27 cycles), 72° C. for 1minute after the last cycle. The PCR product was analyzed on a 1.2%E-gel to confirm successful amplification of the Y. lipolyticacodon-optimized kudzu isoprene synthase gene.

The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kitfollowing manufacturer's protocol: 1 μl PCR reaction, 1 μl Saltsolution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a totalreaction volume of 6 μl. The reaction was incubated at room temperaturefor 5 minutes. One microliter of TOPO reaction was transformed intoTOP10 chemically competent E. coli cells. The transformants wereselected on LA and 50 μg/ml kanamycin plates. Several colonies werepicked and each was inoculated into a 5 ml tube containing LB and 50μg/ml kanamycin and the cultures grown overnight at 37° C. with shakingat 200 rpm. Plasmids were isolated from the overnight culture tubesusing QIAprep Spin Miniprep Kit, following manufacturer's protocol.Several plasmids were sequenced to verify that the DNA sequence wascorrect.

A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimizedkudzu isoprene synthase gene, was used for Gateway Cloning into acustom-made pTrex3g vector. Construction of pTrex3g is described in WO2005/001036 A2. The reaction was performed following manufacturer'sprotocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μlY. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPOdonor vector, 1 μl pTrex3g destination vector, 6 μl TE buffer, pH 8.0 ina total reaction volume of 8 μl. The reaction was incubated at roomtemperature for 1 hour and then 1 μl proteinase K solution was added andthe incubation continued at 37° C. for 10 minutes. Then 1 μl of reactionwas transformed into TOP10 chemically competent E. coli cells. Thetransformants were selected on LA and 50 μg/ml carbenicillin plates.Several colonies were picked and each was inoculated into a 5 ml tubecontaining LB and 50 μg/ml carbenicillin and the cultures were grownovernight at 37° C. with shaking at 200 rpm. Plasmids were isolated fromthe overnight culture tubes using QIAprep Spin Miniprep Kit (Qiagen,Inc.), following manufacturer's protocol. Several plasmids weresequenced to verify that the DNA sequence was correct.

Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprenesynthase pTrex3g plasmid (FIG. 14) into a quad delete Trichoderma reeseistrain was performed using the Biolistic PDS-1000/HE Particle DeliverySystem (see WO 2005/001036 A2). Isolation of stable transformants andshake flask evaluation was performed using protocol listed in Example 11of patent publication WO 2005/001036 A2.

II. Production of Isoprene in Recombinant Strains of T. reesei

One ml of 15 and 36 hour old cultures of isoprene synthase transformantsdescribed above were transferred to head space vials. The vials weresealed and incubated for 5 hours at 30° C. Head space gas was measuredand isoprene was identified by the method described in Example 1. Two ofthe transformants showed traces of isoprene. The amount of isoprenecould be increased by a 14 hour incubation. The two positive samplesshowed isoprene at levels of about 0.5 μg/L for the 14 hour incubation.The untransformed control showed no detectable levels of isoprene. Thisexperiment shows that T. reesei is capable of producing isoprene fromendogenous precursor when supplied with an exogenous isoprene synthase.

Example 6 Production of Isoprene in Yarrowia

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Yarrowia lipolytica.

The starting point for the construction of vectors for the expression ofthe kudzu isoprene synthase gene in Yarrowia lipolytica was the vectorpSPZ1(MAP29spb). The complete sequence of this vector (SEQ ID No:11) isshown in FIG. 15.

The following fragments were amplified by PCR using chromosomal DNA of aY. lipolytica strain GICC 120285 as the template: a promotorless form ofthe URA3 gene, a fragment of 18S ribosomal RNA gene, a transcriptionterminator of the Y. lipolytica XPR2 gene and two DNA fragmentscontaining the promoters of XPR2 and ICL1 genes. The following PCRprimers were used:

ICL1 3 (SEQ ID NO: 69)5′-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCAGG TGAC ICL1 5(SEQ ID NO: 70) 5′-GCAGGTGGGAAACTATGCACTCC XPR 3 (SEQ ID NO: 71)5′-CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG XPR 5 (SEQ ID NO: 72)5′-GGTGTCGACGTACGGTCGAGCTTATTGACC XPRT3 (SEQ ID NO: 73)5′-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG XPRT 5 (SEQ ID NO: 74)5′-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG Y18S3 (SEQ ID NO: 75)5′-GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG Y18S 5 (SEQ ID NO: 76)5′-GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG YURA3 (SEQ ID NO: 77)5′-GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG YURA 50 (SEQ ID NO: 78)5′-GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG YURA 51 (SEQ ID NO: 79)5′-GCGGCCGCAGACTAAATTTATTTCAGTCTCC

For PCR amplification the PfuUltraII polymerase (Stratagene),supplier-provided buffer and dNTPs, 2.5 μM primers and the indicatedtemplate DNA were used in accordance with the manufacturer'sinstructions. The amplification was done using the following cycle: 95°C. for 1 min; 34× (95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3min) and 10 min at 72° C. followed by a 4° C. incubation.

Synthetic DNA molecules encoding the kudzu isoprene synthase gene,codon-optimized for expression in Yarrowia, was obtained from DNA 2.0(FIG. 16; SEQ ID NO:12). Full detail of the construction scheme of theplasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprenesynthase gene under control of XPR2 and ICL1 promoters respectively ispresented in FIG. 18. Control plasmids in which a mating factor gene(MAP29) is inserted in place of an isoprene synthase gene were alsoconstructed (FIGS. 18E and 18F).

A similar cloning procedure can be used to express a poplar (Populusalba x Populus tremula) isoprene synthase gene. The sequence of thepoplar isoprene is described in Miller B. et al. (2001) Planta 213,483-487 and shown in FIG. 17 (SEQ ID NO:13). A construction scheme forthe generation the plasmids pYLA(POP1) and pYLI(POP1) carrying syntheticpoplar isoprene synthase gene under control of XPR2 and ICL1 promotersrespectively is presented in FIGS. 18A and B.

II. Production of Isoprene by Recombinant Strains of Y. lipolytica.

Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digestedwith SacII and used to transform the strain Y. lipolytica CLIB 122 by astandard lithium acetate/polyethylene glycol procedure to uridineprototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract,2% peptone, 2% glucose) overnight, were collected by centrifugation(4000 rpm, 10 min), washed once with sterile water and suspended in 0.1M lithium acetate, pH 6.0. Two hundred μl aliquots of the cellsuspension were mixed with linearized plasmid DNA solution (10-20 μg),incubated for 10 minutes at room temperature and mixed with 1 ml of 50%PEG 4000 in the same buffer. The suspensions were further incubated for1 hour at room temperature followed by a 2 minutes heat shock at 42° C.Cells were then plated on SC his leu plates (0.67% yeast nitrogen base,2% glucose, 100 mg/L each of leucine and histidine). Transformantsappeared after 3-4 days of incubation at 30° C.

Three isolates from the pYLA(KZ1) transformation, three isolates fromthe pYLI(KZ1) transformation, two isolates from the pYLA(MAP29)transformation and two isolates from the pYLI(MAP29) transformation weregrown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0)at 30° C. with shaking. Cells from 10 ml of culture were collected bycentrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 mlscrew cap vials. The vials were incubated overnight at room temperaturewith gentle (60 rpm) shaking. Isoprene content in the headspace of thesevials was analyzed by gas chromatography using mass-spectrometricdetector as described in Example 1. All transformants obtained withpYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene(0.5 μg/L to 1 μg/L, FIG. 20). No isoprene was detected in the headspaceof the control strains carrying phytase gene instead of an isoprenesynthase gene.

Example 7 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and idi, or dxs, or idi and dxs

I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, ordxs, or idi and dxs for the Production of Isoprene in E. coli

i) Construction of pTrcKudzuKan

The bla gene of pTrcKudzu (described in Example 1) was replaced with thegene conferring kanamycin resistance. To remove the bla gene, pTrcKudzuwas digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP),heat killed at 65° C., then end-filled with Klenow fragment and dNTPs.The 5 kbp large fragment was purified from an agarose gel and ligated tothe kan^(r) gene which had been PCR amplified from pCR-Blunt-II-TOPOusing primers MCM22 5′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:14) andMCM23 5′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:15), digested withHindIII and PvuI, and end-filled. A transformant carrying a plasmidconferring kanamycin resistance (pTrcKudzuKan) was selected on LAcontaining kanamycin 50 μg/ml.

ii) Construction of pTrcKudzu yIDI Kan

pTrcKudzuKan was digested with PstI, treated with SAP, heat killed andgel purified. It was ligated to a PCR product encoding idi from S.cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:16) and PstI-YIDI 1R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:17); and the template was S.cerevisiae genomic DNA. The PCR product was digested with NsiI and PstIand gel purified prior to ligation. The ligation mixture was transformedinto chemically competent TOP10 cells and selected on LA containing 50μg/ml kanamycin. Several transformants were isolated and sequenced andthe resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 34 and 35).

iii) Construction of pTrcKudzu DXS Kan

Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding dxsfrom E. coli with a synthetic RBS. The primers for PCR were MCM 135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); andthe template was E. coli genomic DNA. The PCR product was digested withNsiI and PstI and gel purified prior to ligation. The resultingtransformation reaction was transformed into TOP10 cells and selected onLA with kanamycin 50 μg/ml. Several transformants were isolated andsequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (FIGS.36 and 37).

iv) Construction of pTrcKudzu-yIDI-dxs (kan)

pTrcKudzu-yIDI(kan) was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding E.coli dxs with a synthetic RBS (primers MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19);template TOP10 cells) which had been digested with NsiI and PstI and gelpurified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (FIGS.21 and 22).

v) Construction of pCL PtrcKudzu

A fragment of DNA containing the promoter, structural gene andterminator from Example 1 above was digested from pTrcKudzu using SspIand gel purified. It was ligated to pCL1920 which had been digested withPvuII, treated with SAP and heat killed. The resulting ligation mixturewas transformed into TOP10 cells and selected in LA containingspectinomycin 50 μg/ml. Several clones were isolated and sequenced andtwo were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insertin opposite orientations (FIGS. 38-41).

vi) Construction of pCL PtrcKudzu yIDI

The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) abovewas ligated into pCL PtrcKudzu which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu yIDI (FIGS. 42 and 43).

vii) Construction of pCL PtrcKudzu DXS

The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) abovewas ligated into pCL PtrcKudzu (A3) which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu DXS (FIGS. 44 and 45).

II. Measurement of Isoprene in Headspace from Cultures Expressing KudzuIsoprene Synthase, idi, and/or dxs at Different Copy Numbers.

Cultures of E. coli BL21(λDE3) previously transformed with plasmidspTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C),pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Culturesof pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCLPtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL. Cultures wereinduced with 400 μM IPTG at time 0 (OD₆₀₀ approximately 0.5) and samplestaken for isoprene headspace measurement (see Example 1). Results areshown in FIG. 23A-23G.

Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS wasgrown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and usedto inoculate shake flasks of TM3 (13.6 g K₂PO₄, 13.6 g KH₂PO₄, 2.0 gMgSO₄*7H₂O), 2.0 g citric acid monohydrate, 0.3 g ferric ammoniumcitrate, 3.2 g (NH₄)₂SO₄, 0.2 g yeast extract, 1.0 ml 1000× ModifiedTrace Metal Solution, adjusted to pH 6.8 and q.s. to H₂O, and filtersterilized) containing 1% glucose. Flasks were incubated at 30° C. untilan OD₆₀₀ of 0.8 was reached, and then induced with 400 μM IPTG. Sampleswere taken at various times after induction and the amount of isoprenein the head space was measured as described in Example 1. Results areshown in FIG. 23H.

III. Production of Isoprene from Biomass in E. Coli/pTrcKudzu yIDI DXS

The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generateisoprene from three types of biomass; bagasse, corn stover and soft woodpulp with glucose as a control. Hydrolysates of the biomass wereprepared by enzymatic hydrolysis (Brown, L. and Torget, R., 1996, NRELstandard assay method Lap-009 “Enzymatic Saccharification ofLignocellulosic Biomass”) and used at a dilution based upon glucoseequivalents. In this example, glucose equivalents were equal to 1%glucose. A single colony from a plate freshly transformed cells of BL21(DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB pluskanamycin (50 μg/ml). The culture was incubated overnight at 25° C. withshaking. The following day the overnight culture was diluted to an OD₆₀₀of 0.05 in 25 ml of TM3 and 0.2% YE and 1% feedstock. The feedstock wascorn stover, bagasse, or softwood pulp. Glucose was used as a positivecontrol and no glucose was used as a negative control. Cultures wereincubated at 30° C. with shaking at 180 rpm. The culture was monitoredfor OD₆₀₀ and when it reached an OD₆₀₀ of ˜0.8, cultures were analyzedat 1 and 3 hours for isoprene production as described in Example 1.Cultures are not induced. All cultures containing added feedstockproduce isoprene equivalent to those of the glucose positive control.Experiments were done in duplicate and are shown in FIG. 46.

IV. Production of Isoprene from Invert Sugar in E. coli/pTrcKudzuIDIDXS

A single colony from a plate freshly transformed cells of BL21(λDE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL of LB andkanamycin (50 μg/ml). The culture was incubated overnight at 25° C. withshaking. The following day the overnight culture was diluted to an OD₆₀₀of 0.05 in 25 ml of TM3 and 0.2% YE and 1% feedstock. Feedstock wasglucose, inverted glucose or corn stover. The invert sugar feedstock(Danisco Invert Sugar) was prepared by enzymatically treating sucrosesyrup. AFEX corn stover was prepared as described below (Part V). Thecells were grown at 30° C. and the first sample was measured when thecultures reached an OD₆₀₀˜0.8-1.0 (0 hour). The cultures were analyzedfor growth as measured by OD₆₀₀ and for isoprene production as inExample 1 at 0, 1 and 3 hours. Results are shown in FIG. 47.

V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover

AFEX pretreated corn stover was obtained from Michigan BiotechnologyInstitute. The pretreatment conditions were 60% moisture, 1:1 ammonialoading, and 90° C. for 30 minutes, then air dried. The moisture contentin the AFEX pretreated corn stover was 21.27%. The contents of glucanand xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (drybasis), respectively. The saccharification process was as follows; 20 gof AFEX pretreated corn stover was added into a 500 ml flask with 5 mlof 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000, 0.1 mlof Grindamyl H121 (Danisco xylanase product from Aspergillus niger forbread-making industry), and 72.65 ml of DI water. The flask was put inan orbital shaker and incubated at 50° C. for 96 hours. One sample wastaken from the shaker and analyzed using HPLC. The hydrolysate contained38.5 g/l of glucose, 21.8 g/l of xylose, and 10.3 g/l of oligomers ofglucose and/or xylose.

VI. The Effect of Yeast Extract on Isoprene Production in E. coli Grownin Fed-Batch Culture

Fermentation was performed at the 14-L scale as previously describedwith E. coli cells containing the pTrcKudzu yIDI DXS plasmid describedabove. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed atan exponential rate. The total amount of yeast extract delivered to thefermentor was varied between 70-830 g during the 40 hour fermentation.Optical density of the fermentation broth was measured at a wavelengthof 550 nm. The final optical density within the fermentors wasproportional to the amount of yeast extract added (FIG. 48A). Theisoprene level in the off-gas from the fermentor was determined aspreviously described. The isoprene titer increased over the course ofthe fermentation (FIG. 48B). The amount of isoprene produced waslinearly proportional to the amount of fed yeast extract (FIG. 48C).

VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI

A 500 liter fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21(λDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels ofisoprene varied from 50 to 300 μg/L over a time period of 15 hours. Onthe basis of the average isoprene concentrations, the average flowthrough the device and the extent of isoprene breakthrough, the amountof isoprene collected was calculated to be approximately 17 g.

VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown inFed-Batch Culture

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium gas (NH₃) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g,and antibiotic were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in DI H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with0.22 micron filter.

Fermentation was performed in a 500-L bioreactor with E. coli cellscontaining the pTrcKudzu yIDI DXS plasmid. This experiment was carriedout to monitor isoprene formation from glucose and yeast extract at thedesired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was prepared in soytone-yeastextract-glucose medium. After the inoculum grew to OD 0.15, measured at550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-Lsoytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.

Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose werefed at exponential rates. The total amount of glucose and yeast extractdelivered to the bioreactor during the 50 hour fermentation was 181.2 kgand 17.6 kg, respectively. The optical density within the bioreactorover time is shown in FIG. 49A. The isoprene level in the off-gas fromthe bioreactor was determined as previously described. The isoprenetiter increased over the course of the fermentation (FIG. 49B). Thetotal amount of isoprene produced during the 50 hour fermentation was55.1 g and the time course of production is shown in FIG. 49C.

Example 8 Production of Isoprene in E. Coli Expressing Kudzu IsopreneSynthase and Recombinant Mevalonic Acid Pathway Genes

I. Cloning the Lower MVA Pathway

The strategy for cloning the lower mevalonic pathway was as follows.Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase(MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase(MVD) and isopentenyl diphosphate isomerase genes were amplified by PCRfrom S. cerevisiae chromosomal DNA and cloned individually into the pCRBluntII TOPO plasmid (Invitrogen). In some cases, the idi gene wasamplified from E. coli chromosomal DNA. The primers were designed suchthat an E. coli consensus RBS (AGGAGGT (SEQ ID NO:80) or AAGGAGG (SEQ IDNO:81)) was inserted at the 5′ end, 8 bp upstream of the start codon anda PstI site was added at the 3′ end. The genes were then cloned one byone into the pTrcHis2B vector until the entire pathway was assembled.

Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC204508D). The MVK gene was amplified from the chromosome of S.cerevisiae using primers MVKF(5′-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID NO:21) andMVK-Pst1-R (5′-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG, SEQID NO:22) using PfuTurbo as per manufacturer's instructions. The correctsized PCR product (1370 bp) was identified by electrophoresis through a1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resultingplasmid was designated pMVK1. The plasmid pMVK1 was digested with SacIand Taq1 restriction endonucleases and the fragment was gel purified andligated into pTrcHis2B digested with SacI and BstBI. The resultingplasmid was named pTrcMVK1.

The second gene in the mevalonic acid biosynthesis pathway, PMK, wasamplified by PCR using primers: PstI-PMK1 R (5′-GAATTCGCCCTTCTGCAGCTACC,SEQ ID NO:23) and BsiHKA I-PMK1 F(5′-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:24). The PCRreaction was performed using Pfu Turbo polymerase (Stratagene) as permanufacturer's instructions. The correct sized product (1387 bp) wasdigested with PstI and BsiHKI and ligated into pTrcMVK1 digested withPstI. The resulting plasmid was named pTrcKK. The MVD and the idi geneswere cloned in the same manner. PCR was carried out using the primerpairs PstI-MVD 1 R (5′-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID NO:25) andNsiI-MVD 1 F (5′-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID NO:26) toamplify the MVD gene and PstI-YIDI 1 R (5′-CCTTCTGCAGGACGCGTTGTTATAGC,SEQ ID NO:27) and NsiI-YIDI 1 F(5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:16) to amplify theyIDI gene. In some cases the IPP isomerase gene, idi from E. coli wasused. To amplify idi from E. coli chromosomal DNA, the following primerset was used: PstI-CIDI 1 R (5′-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:29)and NsiI-CIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ IDNO:30). Template DNA was chromosomal DNA isolated by standard methodsfrom E. coli FM5 (WO 96/35796 and WO 2004/033646, which are each herebyincorporated by reference in their entireties, particularly with respectto isolation of nucleic acids). The final plasmids were named pKKDIy forthe construct encoding the yeast idi gene or pKKDIc for the constructencoding the E. coli idi gene. The plasmids were transformed into E.coli hosts BL21 for subsequent analysis. In some cases the isoprenesynthase from kudzu was cloned into pKKDIy yielding plasmid pKKDIyIS.

The lower MVA pathway was also cloned into pTrc containing a kanamycinantibiotic resistance marker. The plasmid pTrcKKDIy was digested withrestriction endonucleases ApaI and PstI, the 5930 bp fragment wasseparated on a 1.2% agarose E-gel and purified using the Qiagen GelPurification kit according to the manufacturer's instructions. Theplasmid pTrcKudzuKan, described in Example 7, was digested withrestriction endonucleases ApaI and PstI, and the 3338 bp fragmentcontaining the vector was purified from a 1.2% E-gel using the QiagenGel Purification kit. The 3338 bp vector fragment and the 5930 bp lowerMVA pathway fragment were ligated using the Roche Quick Ligation kit.The ligation mix was transformed into E. coli TOP10 cells andtransformants were grown at 37° C. overnight with selection on LAcontaining kanamycin (50 μg/ml). The transformants were verified byrestriction enzyme digestion and one was frozen as a stock. The plasmidwas designated pTrcKanKKDIy.

II. Cloning a Kudzu Isoprene Synthase Gene into pTrcKanKKDIy

The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu,described in Example 1, using primers MCM505′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAATTTAC T (SEQ IDNO:31) and MCM53 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:4). The resulting PCR fragment was cloned into pCR2.1 and transformedinto E. coli TOP10. This fragment contains the coding sequence for kudzuisoprene synthase and an upstream region containing a RBS from E. coli.Transformants were incubated overnight at 37° C. with selection on LAcontaining carbenicillin (50 μg/ml). The correct insertion of thefragment was verified by sequencing and this strain was designatedMCM93.

The plasmid from strain MCM93 was digested with restrictionendonucleases NsiI and PstI to liberate a 1724 bp insert containing theRBS and kudzu isoprene synthase. The 1724 bp fragment was separated on a1.2% agarose E-gel and purified using the Qiagen Gel Purification kitaccording to the manufacturer's instructions. Plasmid pTrcKanKKDIy wasdigested with the restriction endonuclease PstI, treated with SAP for 30minutes at 37° C. and purified using the Qiagen PCR cleanup kit. Theplasmid and kudzu isoprene synthase encoding DNA fragment were ligatedusing the Roche Quick Ligation kit. The ligation mix was transformedinto E. coli TOP10 cells and transformants were grown overnight at 37°C. with selection on LA containing Kanamycin at 50 μg/ml. The correcttransformant was verified by restriction digestion and the plasmid wasdesignated pTrcKKDyIkISKan (FIGS. 24 and 25). This plasmid wastransformed into BL21(λDE3) cells (Invitrogen).

III. Isoprene Production from Mevalonate in E. coli Expressing theRecombinant Lower Mevalonate Pathway and Isoprene Synthase from Kudzu.

Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt etal., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 andsupplemented with 0.5% glucose and 0.5% mevalonic acid. A controlculture was also set up using identical conditions but without theaddition of 0.5% mevalonic acid. The culture was started from anovernight seed culture with a 1% inoculum and induced with 500 μM IPTGwhen the culture had reached an OD₆₀₀ of 0.3 to 0.5. The cultures weregrown at 30° C. with shaking at 250 rpm. The production of isoprene wasanalyzed 3 hours after induction by using the head space assay describedin Example 1. Maximum production of isoprene was 6.67×10⁴nmol/L_(broth)/OD₆₀₀/hr where L_(broth) is the volume of broth andincludes both the volume of the cell medium and the volume of the cells.The control culture not supplemented with mevalonic acid did not producemeasurable isoprene.

IV. Cloning the Upper MVA Pathway

The upper mevalonate biosynthetic pathway, comprising two genes encodingthree enzymatic activities, was cloned from Enterococcus faecalis. ThemvaE gene encodes a protein with the enzymatic activities of bothacetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, the first and third proteins in the pathway, andthe mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase.The mvaE gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with an E. coli ribosome binding site and a spacer in frontusing the following primers:

CF 07-60 (+) Start of mvaE w/ RBS + ATG start codon SacI (SEQ ID NO: 34)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATT ATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 35)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTC TTAAATC

The mvaS gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with a RBS and spacer from E. coli in front using thefollowing primers:

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between  (SEQ ID NO: 36)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGA TTGATAAACF 07-102 (−) End of mvaS gene BgIII (SEQ ID NO: 37)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The PCR fragments were fused together with PCR using the followingprimers:

CF 07-60 (+) Start of mvaE w/ RBS + ATG start codon SacI (SEQ ID NO: 34)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATTA TTGCF 07-102 (−) End of mvaS gene BgIII (SEQ ID NO: 37)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes SacI and BglII. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated into the commerciallyavailable vector pTrcHis2A, which had been digested with SacI and BglIIand gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA and 50 μg/ml carbenicillin plates. A total of sixcolonies were chosen and grown overnight in LB and 50 μg/mlcarbenicillin and plasmids were isolated using a Qiagen kit. Theplasmids were digested with SacI and BglII to check for inserts and onecorrect plasmid was sequenced with the following primers:

CF 07-58 (+) Start of mvaE gene (SEQ ID NO: 38)5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene(SEQ ID NO: 39) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGCCF 07-82 (+) Start of mvaS gene (SEQ ID NO: 40)5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene(SEQ ID NO: 41) 5′-TTAGTTTCGATAAGAACGAACGGTCF 07-86 (+) Sequence in mvaE (SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATCCF 07-87 (+) Sequence in mvaE (SEQ ID NO: 43)5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE(SEQ ID NO: 44) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS(SEQ ID NO: 45) 5′-GAAACCTACATCCAATCTTTTGCCC

The plasmid called pTrcHis2AUpperPathway#1 was correct by sequencing andwas transformed into the commercially available E. coli strain BL21.Selection was done on LA and 50 μg/ml carbenicillin. Two transformantswere chosen and grown in LB and 50 μg/ml carbenicillin until theyreached an OD₆₀₀ of 1.5. Both strains were frozen in a vial at −80° C.in the presence of glycerol. Strains were designated CF 449 forpTrcHis2AUpperPathway#1 in BL21, isolate #1 and CF 450 forpTrcHis2AUpperPathway#1 in BL21, isolate #2. Both clones were found tobehave identically when analyzed.

V. Cloning of UpperMVA Pathway into pCL1920

The plasmid pTrcHis2AUpperPathway was digested with the restrictionendonuclease SspI to release a fragment containing pTrc-mvaE-mvaS-(Histag)-terminator. In this fragment, the his-tag was not translated. Thisblunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using theQiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbpfragment from pCL1920 was prepared by digesting the vector with therestriction endonuclease PvuII, treating with SAP and gel purifying froma 1.2% E-gel using the Qiagen Gel Purification kit. The two fragmentswere ligated using the Roche Quick Ligation Kit and transformed intoTOP10 chemically competent cells. Transformants were selected on LAcontaining spectinomycin (50 μg/ml). A correct colony was identified byscreening for the presence of the insert by PCR. The plasmid wasdesignated pCL PtrcUpperPathway (FIGS. 26 and 27).

VI. Strains Expressing the Combined Upper and Lower Mevalonic AcidPathways

To obtain a strain with a complete mevalonic acid pathway plus kudzuisoprene synthase, plasmids pTrcKKDyIkISkan and pCLpTrcUpperPathway wereboth transformed into BL21(λDE3) competent cells (Invitrogen) andtransformants were selected on LA containing kanamycin (50 μg/ml) andSpectinomycin (50 μg/ml). The transformants were checked by plasmid prepto ensure that both plasmids were retained in the host. The strain wasdesignated MCM127.

VII. Production of Mevalonic Acid from Glucose in E. coli/pUpperpathway

Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/ppTrcHis2A-mvaE/mvaS are inoculated into LB and carbenicillin (100 μg/ml)and are grown overnight at 37° C. with shaking at 200 rpm. Thesecultures were diluted into 50 ml medium in 250 ml baffled flasks to anOD₆₀₀ of 0.1. The medium was TM3, 1 or 2% glucose, carbenicillin (100μg/ml) or TM3, 1% glucose. hydrolyzed soy oil, and carbenicillin (100μg/ml) or TM3 and biomass (prepared bagasse, corn stover orswitchgrass). Cultures were grown at 30° C. with shaking at 200 rpm forapproximately 2-3 hours until an OD₆₀₀ of 0.4 was reached. At this pointthe expression from the mvaE mvaS construct was induced by the additionof IPTG (400 μM). Cultures were incubated for a further 20 or 40 hourswith samples taken at 2 hour intervals to 6 hour post induction and thenat 24, 36 and 48 hours as needed. Sampling was done by removing 1 ml ofculture, measuring the OD₆₀₀, pelleting the cells in a microfuge,removing the supernatant and analyzing it for mevalonic acid.

A 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid with TM3medium and 2% glucose as the cell medium. A shake flask of these cellsproduced 2-4 grams of mevalonic acid per liter with LB medium and 1%glucose as the cell culture medium. The production of mevalonic acid inthese strains indicated that the MVA pathway was functional in E. coli.

VIII. Production of Isoprene from E. Coli BL21 Containing the Upper andLower MVA Pathway Plus Kudzu Isoprene Synthase.

The following strains were created by transforming in variouscombinations of plasmids containing the upper and lower MVA pathway andthe kudzu isoprene synthase gene as described above and the plasmidscontaining the idi, dxs, and dxr and isoprene synthase genes describedin Example 7. The host cells used were chemically competent BL21(λDE3)and the transformations were done by standard methods. Transformantswere selected on L agar containing kanamycin (50 μg/ml) or kanamycinplus spectinomycin (both at a concentration of 50 μg/ml). Plates weregrown at 37° C. The resulting strains were designated as follows:

Grown on Kanamycin plus Spectinomycin (50 μg/ml each)

MCM127—pCL Upper MVA and pTrcKKDyIkIS (kan) in BL21(λDE3)

MCM131—pCL1920 and pTrcKKDyIkIS (kan) in BL21(λDE3)

MCM125—pCL Upper MVA and pTrcHis2B (kan) in BL21(λDE3)

Grown on Kanamycin (50 μg/ml)

MCM64—pTrcKudzu yIDI DXS (kan) in BL21(λDE3)

MCM50—pTrcKudzu (kan) in BL21(λDE3)

MCM123—pTrcKudzu yIDI DXS DXR (kan) in BL21(λDE3)

The above strains were streaked from freezer stocks to LA andappropriate antibiotic and grown overnight at 37° C. A single colonyfrom each plate was used to inoculate shake flasks (25 ml LB and theappropriate antibiotic). The flasks were incubated at 22° C. overnightwith shaking at 200 rpm. The next morning the flasks were transferred toa 37° C. incubator and grown for a further 4.5 hours with shaking at 200rpm. The 25 ml cultures were centrifuged to pellet the cells and thecells were resuspended in 5 ml LB and the appropriate antibiotic. Thecultures were then diluted into 25 ml LB, % glucose, and the appropriateantibiotic to an OD₆₀₀ of 0.1. Two flasks for each strain were set up,one set for induction with IPTG (800 μM) the second set was not induced.The cultures were incubated at 37° C. with shaking at 250 rpm. One setof the cultures were induced after 1.50 hours (immediately followingsampling time point 1). At each sampling time point, the OD₆₀₀ wasmeasured and the amount of isoprene determined as described inExample 1. Results are presented in Table 3. The amount of isoprene madeis presented as the amount at the peak production for the particularstrain.

TABLE 3 Production of isoprene in E. coli strains Strain Isoprene(μg/L_(broth)/hr/OD MCM50 23.8 MCM64 289 MCM125 ND MCM131 Trace MCM127874 ND: not detected Trace: peak present but not integrable.IX. Analysis of Mevalonic Acid

Mevalonolactone (1.0 g, 7.7 mmol) (CAS#503-48-0) was supplied fromSigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL)and was treated with potassium hydroxide (7.7 mmol) in order to generatethe potassium salt of mevalonic acid. The conversion to mevalonic acidwas confirmed by ¹H NMR analysis. Samples for HPLC analysis wereprepared by centrifugation at 14,000 rpm for 5 minutes to remove cells,followed by the addition of a 300 μl aliquot of supernatant to 900 μl ofH₂O. Perchloric acid (36 μl of a 70% solution) was then added followedby mixing and cooling on ice for 5 minutes. The samples were thencentrifuged again (14,000 rpm for 5 min) and the supernatant transferredto HPLC. Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) wereprepared in the same fashion. Analysis of mevalonic acid (20 μLinjection volume) was performed by HPLC using a BioRad Aminex 87-H+column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6 mL/minwith refractive index (RI) detection. Under these conditions mevalonicacid eluted as the lactone form at 18.5 minutes.

X. Production of Isoprene from E. coli BL21 Containing the Upper MVAPathway Plus Kudzu Isoprene Synthase

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells in fed-batch culture. This experiment demonstrates thatgrowing cells under glucose limiting conditions resulted in theproduction of 2.2 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperPathway (FIG. 26) and pTrcKKDyIkISplasmids. This experiment was carried out to monitor isoprene formationfrom glucose at the desired fermentation pH 7.0 and temperature 30° C.An inoculum of E. coli strain taken from a frozen vial was streaked ontoan LB broth agar plate (with antibiotics) and incubated at 37° C. Asingle colony was inoculated into soytone-yeast extract-glucose medium.After the inoculum grew to OD 1.0 when measured at 550 nm, 500 mL wasused to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 54 hour fermentation was 3.7 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 25 μM when the optical density at 550nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to50 μM when OD₅₅₀ reached 190. IPTG concentration was raised to 100 μM at38 hours of fermentation. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 54. The isoprene level in the off gas from thebioreactor was determined as described herein. The isoprene titerincreased over the course of the fermentation to a final value of 2.2g/L (FIG. 55). The total amount of isoprene produced during the 54 hourfermentation was 15.9 g, and the time course of production is shown inFIG. 56.

XI. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and grown in fed-batch culture at the 15-L scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells in fed-batch culture. This experiment demonstrates thatgrowing cells under glucose limiting conditions resulted in theproduction of 3.0 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× Modified Trace Metal Solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-Lbioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 59 hour fermentation was 2.2 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 50 μM when OD₅₅₀ reached 190. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 93. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 3.0 g/L (FIG. 94). The total amount ofisoprene produced during the 59 hour fermentation was 22.8 g, and thetime course of production is shown in FIG. 95. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 2.2%. The weight percent yield of isoprene from glucose was 1.0%.

XII. Isoprene Fermentation from E. Coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides, Pueraria lobata isoprene synthase, and Kudzu isoprenesynthase was used to produce isoprene from cells in fed-batch culture.This experiment demonstrates that growing cells under glucose limitingconditions resulted in the production of 3.3 g/L of isoprene.

i) Construction of pCLPtrcUpperPathwayHGS2

The gene encoding isoprene synthase from Pueraria lobata wasPCR-amplified using primers NsiI-RBS-HGS F(CTTGATGCATCCTGCATTCGCCCTTAGGAGG, SEQ ID NO:88) and pTrcR(CCAGGCAAATTCTGTTTTATCAG, SEQ ID NO:89), and pTrcKKDyIkIS as a template.The PCR product thus obtained was restriction-digested with NsiI andPstI and gel-purified. The plasmid pCL PtrcUpperPathway wasrestriction-digested with PstI and dephosphorylated using rAPid alkalinephosphatase (Roche) according to manufacturer's instructions.

These DNA fragments were ligated together and the ligation reaction wastransformed into E. coli Top10 chemically competent cells (Invitrogen),plated on L agar containing spectinomycin (50 μg/ml) and incubatedovernight at 370 C. Plasmid DNA was prepared from 6 clones using theQiaquick Spin Mini-prep kit. The plasmid DNA was digested withrestriction enzymes EcoRV and MluI to identify a clone in which theinsert had the right orientation (i.e., the gene oriented in the sameway as the pTrc promoter).

The resulting correct plasmid was designated pCLPtrcUpperPathwayHGS2.This plasmid was assayed using the headspace assay described herein andfound to produce isoprene in E. coli Top10, thus validating thefunctionality of the gene. The plasmid was transformed into BL21(LDE3)containing pTrcKKDyIkIS to yield the strainBL21/pCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS. This strain has an extra copyof the isoprene synthase compared to the BL21/pCL PtrcUpperMVA and pTrcKKDyIkIS strain (Example 8, part XI). This strain also had increasedexpression and activity of HMGS compared to the BL21/pCL PtrcUpperMVAand pTrc KKDyIkIS strain used in Example 8, part XI.

ii) Isoprene Fermentation from E. Coli ExpressingpCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS and Grown in Fed-Batch Culture atthe 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCLPtrcUpperPathwayHGS2 and pTrc KKDyIkIS plasmids.This experiment was carried out to monitor isoprene formation fromglucose at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 37° C. A singlecolony was inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0 measured at 550 nm, 500 mL was used to inoculatea 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 58 hour fermentation was 2.1 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 9. TheIPTG concentration was raised to 50 μM when OD₅₅₀ reached 170. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 104. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 3.3 g/L (FIG. 105). The total amount ofisoprene produced during the 58 hour fermentation was 24.5 g and thetime course of production is shown in FIG. 106. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 2.5%. The weight percent yield of isoprene from glucose was 1.2%.Analysis showed that the activity of the isoprene synthase was increasedby approximately 3-4 times that compared to BL21 expressing CLPtrcUpperMVA and pTrc KKDyIkIS plasmids (data not shown).

XIII. Chromosomal Integration of the Lower Mevalonate Pathway in E.coli.

A synthetic operon containing mevalonate kinase, mevalonate phosphatekinase, mevalonate pyrophosphate decarboxylase, and the IPP isomerasewas integrated into the chromosome of E. coli. If desired, expressionmay be altered by integrating different promoters 5′ of the operon.

Table 4 lists primers used for this experiment.

TABLE 4 Primers MCM78 attTn7 up rev forgcatgctcgagcggccgcTTTTAATCAAACATCCTGCCAA integration constructCTC (SEQ ID NO: 91) MCM79 attTn7 down rev forgatcgaagggcgatcgTGTCACAGTCTGGCGAAACCG integration construct(SEQ ID NO: 92) MCM88 attTn7 up forw forctgaattctgcagatatcTGTTTTTCCACTCTTCGTTCACTT integration constructT (SEQ ID NO: 93) MCM89 attTn7 down forw fortctagagggcccAAGAAAAATGCCCCGCTTACG  integration construct (SEQ ID NO: 94)MCM104 GI1.2 promoter -Gatcgcggccgcgcccttgacgatgccacatcctgagcaaataattcaaccac MVKtaattgtgagcggataacacaaggaggaaacagctatgtcattaccgttcttaacttc (SEQ ID NO: 95) MCM105 aspA terminator -Gatcgggccccaagaaaaaaggcacgtcatctgacgtgccttttttatttgtaga yIDIcgcgttgttatagcattcta (SEQ ID NO: 96) MCM120 Forward of attTn7:aaagtagccgaagatgacggtttgtcacatggagttggcaggatgtttgattaaaattTn7 homology, GB agcAATTAACCCTCACTAAAGGGCGG  marker homology(SEQ ID NO: 97) MCM127 Rev complement ofAGAGTGTTCACCAAAAATAATAACCTTTCCCGG 1.2 GI: GB markerTGCAgaagttaagaacggtaatgacatagctgtttcctccttgtgttatccgcthomology(extra long),cacaattagtggttgaattatttgctcaggatgtggcatcgtcaagggcTAAT promoter, RBS, ATGACGACTCACTATAGGGCTCG (SEQ ID NO: 98)i) Target Vector Construction

The attTn7 site was selected for integration. Regions of homologyupstream (attTn7 up) (primers MCM78 and MCM79) and downstream (attTn7down) (primers MCM88 and MCM89) were amplified by PCR from MG1655 cells.A 50 μL reaction with 1 μL 10 μM primers, 3 μL ddH2O, 45 μL InvitrogenPlatinum PCR Supermix High Fidelity, and a scraped colony of MG1655 wasdenatured for 2:00 at 940 C, cycled 25 times (2:00 at 940 C, 0:30 at 500C, and 1:00 at 680 C), extended for 7:00 at 720 C, and cooled to 40 C.This resulting DNA was cloned into pCR2.1 (Invitrogen) according to themanufacturer's instructions, resulting in plasmids MCM278 (attTn7 up)and MCM252 (attTn7 down). The 832 bp ApaI-PvuI fragment digested and gelpurified from MCM252 was cloned into ApaI-PvuI digested and gel purifiedplasmid pR6K, creating plasmid MCM276. The 825 bp PstI-NotI fragmentdigested and gel purified from MCM278 was cloned into PstI-NotI digestedand gel purified MCM276, creating plasmid MCM281.

ii) Cloning of Lower Pathway and Promoter

MVK-PMK-MVD-IDI genes were amplified from pTrcKKDyIkIS with primersMCM104 and MCM105 using Roche Expand Long PCR System according to themanufacturer's instructions. This product was digested with NotI andApaI and cloned into MCM281 which had been digested with NotI and ApaIand gel purified. Primers MCM120 and MCM127 were used to amplify CMRcassette from the GeneBridges FRT-gb2-Cm-FRT template DNA usingStratagene Pfu Ultra II. A PCR program of denaturing at 950 C for 4:00,5 cycles of 950 C for 0:20, 550 C for 0:20, 720 C for 2:00, 25 cycles of950 C for 0:20, 580 C for 0:20, 720 C for 2:00, 720 C for 10:00, andthen cooling to 40 C was used with four 50 μL PCR reactions containing 1μL˜10 ng/μL template, 1 μL each primer, 1.25 μL 10 mM dNTPs, 5 μL 10×buffer, 1 μL enzyme, and 39.75 μL ddH₂O. Reactions were pooled, purifiedon a Qiagen PCR cleanup column, and used to electroporate water-washedPir1 cells containing plasmid MCM296. Electroporation was carried out in2 mM cuvettes at 2.5V and 200 ohms. Electroporation reactions wererecovered in LB for 3 hr at 300 C. Transformant MCM330 was selected onLA with CMP5, Kan50 (FIGS. 107 and 108A-108C).

iii) Integration into E. coli Chromosome

Miniprepped DNA (Qiaquick Spin kit) from MCM330 was digested with SnaBIand used to electroporate BL21(DE3) (Novagen) or MG1655 containingGeneBridges plasmid pRedET Carb. Cells were grown at 300 C to ˜OD1 theninduced with 0.4% L-arabinose at 370 C for 1.5 hours. These cells werewashed three times in 40 C ddH20 before electroporation with 2 μL ofDNA. Integrants were selected on L agar with containing chloramphenicol(5 μg/ml) and subsequently confirmed to not grow on L agar+Kanamycin (50μg/ml). BL21 integrant MCM331 and MG1655 integrant MCM333 were frozen.

iv) Construction of pET24D-Kudzu Encoding Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was subcloned into the pET24d vector(Novagen) from the pCR2.1 vector (Invitrogen). In particular, the kudzuisoprene synthase gene was amplified from the pTrcKudzu template DNAusing primers MCM50 5′-GATCATGCAT TCGCCCTTAG GAGGTAAAAA AACATGTGTGCGACCTCTTC TCAATTTACT (SEQ ID NO:99) and MCM53 5′-CGGTCGACGG ATCCCTGCAGTTAGACATAC ATCAGCTG (SEQ ID NO:4). PCR reactions were carried out usingTaq DNA Polymerase (Invitrogen), and the resulting PCR product wascloned into pCR2.1-TOPO TA cloning vector (Invitrogen), and transformedinto E. coli Top 10 chemically competent cells (Invitrogen).Transformants were plated on L agar containing carbenicillin (50 μg/ml)and incubated overnight at 37° C. Five ml Luria Broth culturescontaining carbenicillin 50 μg/ml were inoculated with singletransformants and grown overnight at 37° C. Five colonies were screenedfor the correct insert by sequencing of plasmid DNA isolated from 1 mlof liquid culture (Luria Broth) and purified using the QIAprep SpinMini-prep Kit (Qiagen). The resulting plasmid, designated MCM93,contains the kudzu isoprene synthase coding sequence in a pCR2.1backbone.

The kudzu coding sequence was removed by restriction endonucleasedigestion with PciI and BamH1 (Roche) and gel purified using theQIAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digestedwith NcoI and BamHI (Roche), treated with shrimp alkaline phosphatase(Roche), and purified using the QIAprep Spin Mini-prep Kit (Qiagen). Thekudzu isoprene synthase fragment was ligated to the NcoI/BamH1 digestedpET24d using the Rapid DNA Ligation Kit (Roche) at a 5:1 fragment tovector ratio in a total volume of 20 μl. A portion of the ligationmixture (5 μl) was transformed into E. coli Top 10 chemically competentcells and plated on L agar containing kanamycin (50 μg/ml). The correcttransformant was confirmed by sequencing and transformed into chemicallycompetent BL21(λDE3) pLysS cells (Novagen). A single colony was selectedafter overnight growth at 37° C. on L agar containing kanamycin (50μg/ml). A map of the resulting plasmid designated as pET24D-Kudzu isshown in FIG. 109. The sequence of pET24D-Kudzu (SEQ ID NO:101) is shownin FIGS. 110A and 110B. Isoprene synthase activity was confirmed using aheadspace assay.

v) Production Strains

Strains MCM331 and MCM333 were cotransformed with plasmidspCLPtrcupperpathway and either pTrcKudzu or pETKudzu, resulting in thestrains shown in Table 5.

TABLE 5 Production Strains Isoprene Integrated Upper MVA synthaseProduction Background Lower plasmid plasmid Stain BL21(DE3) MCM331pCLPtrcUpper pTrcKudzu MCM343 Pathway BL21(DE3) MCM331 pCLPtrcUpperpET24D- MCM335 Pathway Kudzu MG1655 MCM333 pCLPtrcUpper pTrcKudzu MCM345Pathwayvi) Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale.Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH2O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the gi1.2 integrated lower MVA pathway described aboveand the pCL PtrcUpperMVA and pTrcKudzu plasmids. This experiment wascarried out to monitor isoprene formation from glucose at the desiredfermentation pH 7.0 and temperature 30° C. An inoculum of E. coli straintaken from a frozen vial was streaked onto an LB broth agar plate (withantibiotics) and incubated at 37° C. A single colony was inoculated intotryptone-yeast extract medium. After the inoculum grew to OD 1.0,measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 57 hour fermentation was 3.9 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 100 μMwhen the carbon dioxide evolution rate reached 100 mmol/L/hr. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 111A. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 1.6 g/L (FIG. 111B). The specificproductivity of isoprene over the course of the fermentation is shown inFIG. 111C and peaked at 1.2 mg/OD/hr. The total amount of isopreneproduced during the 57 hour fermentation was 16.2 g. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 0.9%. The weight percent yield of isoprene from glucose was 0.4%.

XIV. Production of Isoprene from E. Coli BL21 Containing the KudzuIsoprene Synthase Using Glycerol as a Carbon Source

A 15-L scale fermentation of E. coli expressing Kudzu isoprene synthasewas used to produce isoprene from cells fed glycerol in fed-batchculture. This experiment demonstrates that growing cells in the presenceof glycerol (without glucose) resulted in the production of 2.2 mg/L ofisoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, and 1000× modified tracemetal solution 1 ml. All of the components were added together anddissolved in diH₂O. This solution was autoclaved. The pH was adjusted to7.0 with ammonium hydroxide (30%) and q.s. to volume. Glycerol 5.1 g,thiamine*HCl 0.1 g, and antibiotics were added after sterilization andpH adjustment.

1000× Modified Trace Metal Solution:

The medium was generated using the following components per literfermentation medium: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pTrcKudzu plasmid. This experiment was carried outto monitor isoprene formation from glycerol at the desired fermentationpH 7.0 and temperature 35° C. An inoculum of E. coli strain taken from afrozen vial was streaked onto an LA broth agar plate (with antibiotics)and incubated at 37° C. A single colony was inoculated intosoytone-yeast extract-glucose medium and grown at 35° C. After theinoculum grew to OD 1.0, measured at 550 nm, 600 mL was used toinoculate a 7.5-L bioreactor.

Glycerol was fed at an exponential rate until cells reached an opticaldensity at 550 nm (OD₅₅₀) of 153. The total amount of glycerol deliveredto the bioreactor during the 36 hour fermentation was 1.7 kg. Other thanthe glucose in the inoculum, no glucose was added to the bioreactor.Induction was achieved by adding IPTG. The IPTG concentration wasbrought to 20 μM when the OD₅₅₀ reached a value of 50. The OD₅₅₀ profilewithin the bioreactor over time is shown in FIG. 57. The isoprene levelin the off gas from the bioreactor was determined as described herein.The isoprene titer increased over the course of the fermentation to afinal value of 2.2 mg/L (FIG. 58). The total amount of isoprene producedduring the 54 hour fermentation was 20.9 mg, and the time course ofproduction is shown in FIG. 59.

XV. Isoprene Fermentation from E. Coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L ScaleUsing Invert Sugar as a Carbon Source

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells fed invert sugar in fed-batch culture. This experimentdemonstrates that growing cells in the presence of invert sugar resultedin the production of 2.4 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× Modified Trace Metal Solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Invert sugar 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from invertsugar at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 37° C. A singlecolony was inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0, measured at 550 nm, 500 mL was used toinoculate a 5-L bioreactor.

Invert sugar was fed at an exponential rate until cells reached thestationary phase. After this time the invert sugar feed was decreased tomeet metabolic demands. The total amount of invert sugar delivered tothe bioreactor during the 44 hour fermentation was 2.4 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 9. TheIPTG concentration was raised to 50 μM when OD₅₅₀ reached 200. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 96. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 2.4 g/L (FIG. 97). The total amount ofisoprene produced during the 44 hour fermentation was 18.4 g and thetime course of production is shown in FIG. 98. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 1.7%. The weight percent yield of isoprene from glucose was 0.8%.

Example 9 Construction of the Upper and Lower MVA Pathway forIntegration into Bacillus subtilis

I. Construction of the Upper MVA Pathway in Bacillus subtilis

The upper pathway from Enterococcus faecalis is integrated into B.subtilis under control of the aprE promoter. The upper pathway consistsof two genes; mvaE, which encodes for AACT and HMGR, and mvaS, whichencodes for HMGS. The two genes are fused together with a stop codon inbetween, an RBS site in front of mvaS, and are under the control of theaprE promoter. A terminator is situated after the mvaE gene. Thechloramphenicol resistance marker is cloned after the mvaE gene and theconstruct is integrated at the aprE locus by double cross over usingflanking regions of homology.

Four DNA fragments are amplified by PCR such that they contain overhangsthat will allow them to be fused together by a PCR reaction. PCRamplifications are carried out using Herculase polymerase according tomanufacturer's instructions.

1: PaprE CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-94 (−) Fuse PaprE to mvaE(SEQ ID NO: 83) 5′-CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAATemplate: Bacillus subtilis chromosomal DNA 2: mvaECF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon)(SEQ ID NO: 84) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 35)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTC TTAAATCTemplate: Enterococcus faecalis chromosomal DNA (from ATCC) 3. mvaSCF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 36)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGAT TGATAAACF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: Enterococcus faecalis chromosomal DNA4. B. amyliquefaciens alkaline serine protease terminatorCF 07-123 (+) Fuse the end of mvaS to the terminator (SEQ ID NO: 86)5′-ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCGCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTCTemplate: Bacillus amyliquefaciens chromosomal DNA PCR Fusion Reactions5. Fuse mvaE to mvaS CF 07-93 (+) fuse mvaE to the aprE promoter(GTG start codon) (SEQ ID NO: 84)5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: #2 and 3 from above 6. Fuse mvaE-mvaS to aprE promoterCF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-124 (−) Fuse the end of mvaS to theterminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate #1 and #4 from above 7. Fuse PaprE-mvaE-mvaS to terminatorCF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTC Template: #4 and #6

The product is digested with restriction endonucleases PstI/BamHI andligated to pJM102 (Perego, M. 1993. Integrational vectors for geneticmanipulation in Bacillus subtilis, p. 615-624. In A. L. Sonenshein, J.A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positivebacteria: biochemistry, physiology, and molecular genetics. AmericanSociety for Microbiology, Washington, D.C.) which is digested withPstI/BamHI. The ligation is transformed into E. coli TOP 10 chemicallycompetent cells and transformants are selected on LA containingcarbenicillin (50 μg/ml). The correct plasmid is identified bysequencing and is designated pJMUpperpathway2 (FIGS. 50 and 51).Purified plasmid DNA is transformed into Bacillus subtilis aprEnprEPxyl-comK and transformants are selected on L agar containingchloramphenicol (5 μg/ml). A correct colony is selected and is platedsequentially on L agar containing chloramphenicol 10, 15 and 25 μg/ml toamplify the number of copies of the cassette containing the upperpathway.

The resulting strain is tested for mevalonic acid production by growingin LB containing 1% glucose and 1%. Cultures are analyzed by GC for theproduction of mevalonic acid.

This strain is used subsequently as a host for the integration of thelower mevalonic acid pathway.

The following primers are used to sequence the various constructs above.

Sequencing primers:

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-58 (+) Start of mvaE gene(SEQ ID NO: 38) 5′-ATGAAAACAGTAGTTATTATTGATGCCF 07-59 (−) End of mvaE gene (SEQ ID NO: 39)5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC CF 07-82 (+) Start of mvaS gene(SEQ ID NO: 40) 5′-ATGACAATTGGGATTGATAAAATTAGCF 07-83 (−) End of mvaS gene (SEQ ID NO: 41)5′-TTAGTTTCGATAAGAACGAACGGT CF 07-86 (+) Sequence in mvaE(SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATC CF 07-87 (+) Sequence in mvaE(SEQ ID NO: 43) 5′-TTGCCAATCATATGATTGAAAATCCF 07-88 (+) Sequence in mvaE (SEQ ID NO: 44)5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS (SEQ ID NO: 45)5′-GAAACCTACATCCAATCTTTTGCCC

Transformants are selected on LA containing chloramphenicol at aconcentration of 5 μg/ml. One colony is confirmed to have the correctintegration by sequencing and is plated on LA containing increasingconcentrations of chloramphenicol over several days, to a final level of25 μg/ml. This results in amplification of the cassette containing thegenes of interest. The resulting strain is designated CF 455:pJMupperpathway#1× Bacillus subtilis aprEnprE Pxyl comK (amplified togrow on LA containing chloramphenicol 25 μg/ml).

II. Construction of the Lower MVA Pathway in Bacillus subtilis

The lower MVA pathway, consisting of the genes mvk1, pmk, mpd and idiare combined in a cassette consisting of flanking DNA regions from thenprE region of the B. subtilis chromosome (site of integration), theaprE promoter, and the spectinomycin resistance marker (see FIGS. 28 and29). This cassette is synthesized by DNA2.0 and is integrated into thechromosome of B. subtilis containing the upper MVA pathway integrated atthe aprE locus. The kudzu isoprene synthase gene is expressed from thereplicating plasmid described in Example 4 and is transformed into thestrain with both upper and lower pathways integrated.

Example 10 Exemplary Isoprene Compositions and Methods of Making them

I. Compositional Analysis of Fermentation Off-Gas Containing Isoprene

A 14 L scale fermentation was performed with a recombinant E. coli BL21(DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkISencoding the full mevalonate pathway for isoprenoid precursorbiosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and anisoprene synthase from Kudzu. Fermentation off-gas from the 14 L tankwas collected into 20 mL headspace vials at around the time of peakisoprene productivity (27.9 hours elapsed fermentation time, “EFT”) andanalyzed by headspace GC/MS for volatile components.

Headspace analysis was performed with an Agilent 6890 GC/MS systemfitted with an Agilent HP-5MS GC/MS column (30 m×250 μm; 0.25 μm filmthickness). A combiPAL autoinjector was used for sampling 500 μLaliquots from 20 mL headspace vials. The GC/MS method utilized helium asthe carrier gas at a flow of 1 mL/min. The injection port was held at250° C. with a split ratio of 50:1. The oven temperature was held at 37°C. for an initial 2 minute period, followed an increase to 237° C. at arate of 25° C./min for a total method time of 10 minutes. The Agilent5793N mass selective detector scanned from m/z 29 to m/z 300. The limitof detection of this system is approximately 0.1 μg/L_(gas) orapproximately 0.1 ppm. If desired, more sensitive equipment with a lowerlimit of detection may be used.

The off-gas consisted of 99.925% (v/v) permanent gases (N₂, CO₂ and O₂),approximately 0.075% isoprene (2-methyl-1,3-butadiene) (˜750 ppmv, 2100μg/L) and minor amounts (<50 ppmv) of ethanol, acetone, and two C5prenyl alcohols. The amount of water vapor was not determined but wasestimated to be equal to the equilibrium vapor pressure at 0° C. Thecomposition of the volatile organic fraction was determined byintegration of the area under the peaks in the GC/MS chromatogram (FIGS.86A and 86B) and is listed in Table 6. Calibration curves for ethanoland acetone standards enabled the conversion of GC area to gas phaseconcentration in units of μg/L using standard methods.

TABLE 6 Composition of volatile organic components in fermentationoff-gas. The off-gas was analyzed at the 27.9 hour time point of afermentation using an E. coli BL21 (DE3) strain expressing aheterologous mevalonate pathway, an isoprenyl pyrophosphate isomerasefrom yeast, and an isoprene synthase from Kudzu. Compound RT (min) GCarea Area % Conc. (μg/L) Ethanol 1.669 239005 0.84 62 +/− 6 Acetone1.703 288352 1.02 42 +/− 4 Isoprene (2-methyl- 1.829 27764544 97.81 2000+/− 200 1,3-butadiene) 3-methyl-3-buten-1-ol 3.493 35060 0.12 <103-methyl-2-buten-1-ol 4.116 58153 0.20 <10II. Measurement of Trace Volatile Organic Compounds (VOCs) Co-Producedwith Isoprene During Fermentation of a Recombinant E. coli Strain

A 14 L scale fermentation was performed with a recombinant E. coli BL21(DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkIS)encoding the full mevalonate pathway for isoprenoid precursorbiosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and anisoprene synthase from Kudzu.

Fermentation off-gas was passed through cooled headspace vials in orderto concentrate and identify trace volatile organic components. Theoff-gas from this fermentation was sampled at a rate of 1 L/min for 10minutes through a 20 mL headspace vial packed with quartz wool (2 g) andcooled to −78° C. with dry ice. The vial was recapped with a fresh vialcap and analyzed by headspace GC/MS for trapped VOCs using theconditions described in Example 10, part I. The ratios of compoundsobserved in FIGS. 87A-87D are a combination of overall level in thefermentation off-gas, the relative vapor pressure at −78° C., and thedetector response of the mass spectrometer. For example, the low levelof isoprene relative to oxygenated volatiles (e.g., acetone and ethanol)is a function of the high volatility of this material such that it doesnot accumulate in the headspace vial at −78° C.

The presence of many of these compounds is unique to isoprenecompositions derived from biological sources. The results are depictedin FIGS. 87A-87D and summarized in Tables 7A and 7B.

TABLE 7A Trace volatiles present in off-gas produced by E. coli BL21(DE3) (pCL upperMev; pTrcKKDyIkIS) following cryo-trapping at −78° C. RTArea Ratio Compound (min) GC Area¹ %² %³ Acetaldehyde 1.542 40198614.841 40.14 Ethanol 1.634 10553620 12.708 105.39 Acetone 1.727 72363238.714 72.26 2-methyl-1,3-butadiene 1.777 10013714 12.058 100.001-propanol 1.987 163574 0.197 1.63 Diacetyl 2.156 221078 0.266 2.212-methyl-3-buten-2-ol 2.316 902735 1.087 9.01 2-methyl-1-propanol 2.451446387 0.538 4.46 3-methyl-1-butanal 2.7 165162 0.199 1.65 1-butanol2.791 231738 0.279 2.31 3-methyl-3-buten-1-ol 3.514 14851860 17.884148.32 3-methyl-1-butanol 3.557 8458483 10.185 84.473-methyl-2-buten-1-ol 4.042 18201341 21.917 181.76 3-methyl-2-butenal4.153 1837273 2.212 18.35 3-methylbutyl acetate 5.197 196136 0.236 1.963-methyl-3-buten-1-yl acetate 5.284 652132 0.785 6.51 2-heptanone 5.34867224 0.081 0.67 2,5-dimethylpyrazine 5.591 58029 0.070 0.583-methyl-2-buten-1-yl acetate 5.676 1686507 2.031 16.846-methyl-5-hepten-2-one 6.307 101797 0.123 1.02 2,4,5-trimethylpyridine6.39 68477 0.082 0.68 2,3,5-trimethylpyrazine 6.485 30420 0.037 0.30(E)-3,7-dimethyl-1,3,6-octatriene 6.766 848928 1.022 8.48(Z)-3,7-dimethyl-1,3,6-octatriene 6.864 448810 0.540 4.483-methyl-2-buten-1-yl butyrate 7.294 105356 0.127 1.05 Citronellal 7.756208092 0.251 2.08 2,3-cycloheptenolpyridine 8.98 1119947 1.349 11.18 ¹GCarea is the uncorrected area under the peak corresponding to the listedcompound. ²Area % is the peak area expressed as a % relative to thetotal peak area of all compounds. ³Ratio % is the peak area expressed asa % relative to the peak area of 2-methyl-1,3-butadiene.

TABLE 7B Trace volatiles present in off-gas produced by E. coli BL21(DE3) (pCL upperMev; pTrcKKDyIkIS) following cryo-trapping at −196° C.RT Ratio Compound (min) GC Area¹ Area %² %³ Acetaldehyde 1.54 16557100.276 0.33 Methanethiol 1.584 173620 0.029 0.03 Ethanol 1.631 102596801.707 2.03 Acetone 1.722 73089100 12.164 14.43 2-methyl-1,3-butadiene1.771 506349429 84.269 100.00 methyl acetate 1.852 320112 0.053 0.061-propanol 1.983 156752 0.026 0.03 Diacetyl 2.148 67635 0.011 0.012-butanone 2.216 254364 0.042 0.05 2-methyl-3-buten-2-ol 2.312 6847080.114 0.14 ethyl acetate 2.345 2226391 0.371 0.44 2-methyl-1-propanol2.451 187719 0.031 0.04 3-methyl-1-butanal 2.696 115723 0.019 0.023-methyl-2-butanone 2.751 116861 0.019 0.02 1-butanol 2.792 54555 0.0090.01 2-pentanone 3.034 66520 0.011 0.01 3-methyl-3-buten-1-ol 3.5161123520 0.187 0.22 3-methyl-1-butanol 3.561 572836 0.095 0.11 ethylisobutyrate 3.861 142056 0.024 0.03 3-methyl-2-buten-1-ol 4.048 3025580.050 0.06 3-methyl-2-butenal 4.152 585690 0.097 0.12 butyl acetate4.502 29665 0.005 0.01 3-methylbutyl acetate 5.194 271797 0.045 0.053-methyl-3-buten-1-yl acetate 5.281 705366 0.117 0.143-methyl-2-buten-1-yl acetate 5.675 815186 0.136 0.16 (E)-3,7-dimethyl-6.766 207061 0.034 0.04 1,3,6-octatriene (Z)-3,7-dimethyl- 6.863 942940.016 0.02 1,3,6-octatriene 2,3-cycloheptenolpyridine 8.983 135104 0.0220.03 ¹GC area is the uncorrected area under the peak corresponding tothe listed compound. ²Area % is the peak area expressed as a % relativeto the total peak area of all compounds. ³Ratio % is the peak areaexpressed as a % relative to the peak area of 2-methyl-1,3-butadiene.III. Absence of C5 Hydrocarbon Isomers in Isoprene Derived fromFermentation.

Cryo-trapping of isoprene present in fermentation off-gas was performedusing a 2 mL headspace vial cooled in liquid nitrogen. The off-gas (1L/min) was first passed through a 20 mL vial containing sodium hydroxidepellets in order to minimize the accumulation of ice and solid CO₂ inthe 2 mL vial (−196° C.). Approximately 10 L of off-gas was passedthrough the vial, after which it was allowed to warm to −78° C. withventing, followed by resealing with a fresh vial cap and analysis byGC/MS.

GC/MS headspace analysis was performed with an Agilent 6890 GC/MS systemusing a 100 μL gas tight syringe in headspace mode. A Zebron ZB-624GC/MS column (30 m×250 μm; 1.40 μm film thickness) was used forseparation of analytes. The GC autoinjector was fitted with a gas-tight100 μL syringe, and the needle height was adjusted to allow theinjection of a 50 μL headspace sample from a 2 mL GC vial. The GC/MSmethod utilized helium as the carrier gas at a flow of 1 mL/min. Theinjection port was held at 200° C. with a split ratio of 20:1. The oventemperature was held at 37° C. for the 5 minute duration of theanalysis. The Agilent 5793N mass selective detector was run in singleion monitoring (SIM) mode on m/z 55, 66, 67 and 70. Under theseconditions, isoprene was observed to elute at 2.966 minutes (FIG. 88B).A standard of petroleum derived isoprene (Sigma-Aldrich) was alsoanalyzed using this method and was found to contain additional C5hydrocarbon isomers, which eluted shortly before or after the main peakand were quantified based on corrected GC area (FIG. 88A).

TABLE 8A GC/MS analysis of petroleum-derived isoprene Area % of total C5Compound RT (min) GC area hydrocarbons 2-methyl-1-butene 2.689 18.2 ×10³ 0.017% (Z)-2-pentene 2.835 10.6 × 10⁴ 0.101% Isoprene 2.966 10.4 ×10⁷ 99.869%  1,3-cyclopentadiene 3.297 12.8 × 10³ 0.012% (CPD)

TABLE 8B GC/MS analysis of fermentation-derived isoprene (% total C5hydrocarbons) Corrected GC % of total C5 Compound RT (min) Areahydrocarbons Isoprene 2.966 8.1 × 10⁷ 100%

In a separate experiment, a standard mixture of C5 hydrocarbons wasanalyzed to determine if the detector response was the same for each ofthe compounds. The compounds were 2-methyl-1-butene,2-methyl-1,3-butadiene, (E)-2-pentene, (Z)-2-pentene and(E)-1,3-pentadiene. In this case, the analysis was performed on anAgilent DB-Petro column (100 m×0.25 mm, 0.50 um film thickness) held at50° C. for 15 minutes. The GC/MS method utilized helium as the carriergas at a flow of 1 mL/min. The injection port was held at 200° C. with asplit ratio of 50:1. The Agilent 5793N mass selective detector was runin full scan mode from m/z 19 to m/z 250. Under these conditions, a 100μg/L concentration of each standard produced the same detector responsewithin experimental error.

IV. Compositions Comprising Isoprene Adsorbed to a Solid Phase.

Biologically-produced isoprene was adsorped to activated carbonresulting in a solid phase containing 50 to 99.9% carbon, 0.1% to 50%isoprene, 0.01% to 5% water, and minor amounts (<0.1%) of other volatileorganic components.

Fermentation off-gas was run through a copper condensation coil held at0° C., followed by a granulated silica desiccant filter in order toremove water vapor. The dehumidified off-gas was then run through carboncontaining filters (Koby Jr, Koby Filters, Mass.) to the point at whichbreakthrough of isoprene was detected in the filter exhaust by GC/MS.The amount of isoprene adsorped to the cartridge can be determinedindirectly by calculating the concentration in the off-gas, the overallflow rate and the percent breakthrough over the collection period.Alternately the adsorped isoprene can be recovered from the filters bythermal, vacuum, or solvent-mediated desorption.

V. Collection and Analysis of Condensed Isoprene.

Fermentation off-gas is dehumidified, and the CO₂ removed by filtrationthrough a suitable adsorbant (e.g., ascarite). The resulting off-gasstream is then run through a liquid nitrogen-cooled condenser in orderto condense the VOCs in the stream. The collection vessel containst-butyl catechol to inhibit the resulting isoprene condensate. Thecondensate is analyzed by GC/MS and NMR in order to determine purityusing standard methods, such as those described herein.

VI. Production of Prenyl Alcohols by Fermentation

Analysis of off-gas from an E. coli BL21 (DE3) strain expressing a Kudzuisoprene synthase revealed the presence of both isoprene and3-methyl-3-buten-1-ol (isoprenol). The levels of the two compounds inthe fermentation off-gas over the fermentation are shown in FIG. 89 asdetermined by headspace GC/MS. Levels of isoprenol(3-methyl-3-buten-1-ol, 3-MBA) attained was nearly 10 μg/L_(offgas) inthis experiment. Additional experiments produced levels of approximately20 μg/L_(offgas) in the fermentation off-gas.

Example 11 The De-Coupling of Growth and Production of Isoprene in E.coli Expressing Genes from the Mevalonic Acid Pathway and Fermented in aFed-Batch Culture

Example 11 illustrates the de-coupling of cell growth from mevalonicacid and isoprene production.

I. Fermentation Conditions

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH2O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl2*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed with E. coli cells containing thepTrcHis2AUpperPathway (also called pTrcUpperMVA, FIGS. 91 and 92A-92C)(50 μg/ml carbenicillin) or the pCL PtrcUpperMVA (also called pCLPtrcUpperPathway (FIG. 26)) (50 μg/ml spectinomycin) plasmids. Forexperiments in which isoprene was produced, the E. coli cells alsocontained the pTrc KKDyIkIS (50 μg/ml kanamycin) plasmid. Theseexperiments were carried out to monitor mevalonic acid or isopreneformation from glucose at the desired fermentation pH 7.0 andtemperature 30° C. An inoculum of an E. coli strain taken from a frozenvial was streaked onto an LA broth agar plate (with antibiotics) andincubated at 37° C. A single colony was inoculated into tryptone-yeastextract medium. After the inoculum grew to optical density 1.0 whenmeasured at 550 nm, it was used to inoculate the bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. Induction was achieved by adding IPTG. The mevalonicacid concentration in fermentation broth was determined by applyingperchloric acid (Sigma-Aldrich #244252) treated samples (0.3 M incubatedat 4° C. for 5 minutes) to an organic acids HPLC column (BioRad#125-0140). The concentration was determined by comparing the brothmevalonic acid peak size to a calibration curve generated frommevalonolacetone (Sigma-Aldrich #M4667) treated with perchloric acid toform D,L-mevalonate. The isoprene level in the off gas from thebioreactor was determined as described herein. The isoprene titer isdefined as the amount of isoprene produced per liter of fermentationbroth.

II. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressingthe pTrcUpperMVA Plasmid at a 150-L Scale

BL21 (DE3) cells that were grown on a plate as explained above inExample 11, part I were inoculated into a flask containing 45 mL oftryptone-yeast extract medium and incubated at 30° C. with shaking at170 rpm for 5 hours. This solution was transferred to a 5-L bioreactorof tryptone-yeast extract medium, and the cells were grown at 30° C. and27.5 rpm until the culture reached an OD₅₅₀ of 1.0. The 5 L of inoculumwas seeded into a 150-L bioreactor containing 45-kg of medium. The IPTGconcentration was brought to 1.1 mM when the OD₅₅₀ reached a value of10. The OD₅₅₀ profile within the bioreactor over time is shown in FIG.60A. The mevalonic acid titer increased over the course of thefermentation to a final value of 61.3 g/L (FIG. 60B). The specificproductivity profile throughout the fermentation is shown in FIG. 60Cand a comparison to FIG. 60A illustrates the de-coupling of growth andmevalonic acid production. The total amount of mevalonic acid producedduring the 52.5 hour fermentation was 4.0 kg from 14.1 kg of utilizedglucose. The molar yield of utilized carbon that went into producingmevalonic acid during fermentation was 34.2%.

III. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressingthe pTrcUpperMVA Plasmid at a 15-L Scale

BL21 (DE3) cells that were grown on a plate as explained above inExample 11, part I were inoculated into a flask containing 500 mL oftryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀1.0. This material was seeded into a 15-L bioreactor containing 4.5-kgof medium. The IPTG concentration was brought to 1.0 mM when the OD₅₅₀reached a value of 10. The OD₅₅₀ profile within the bioreactor over timeis shown in FIG. 61A. The mevalonic acid titer increased over the courseof the fermentation to a final value of 53.9 g/L (FIG. 61B). Thespecific productivity profile throughout the fermentation is shown inFIG. 61C and a comparison to FIG. 61A illustrates the de-coupling ofgrowth and mevalonic acid production. The total amount of mevalonic acidproduced during the 46.6 hour fermentation was 491 g from 2.1 kg ofutilized glucose. The molar yield of utilized carbon that went intoproducing mevalonic acid during fermentation was 28.8%.

IV. Mevalonic Acid Production from E. coli FM5 Cells Expressing thepTrcUpperMVA Plasmid at a 15-L Scale

FM5 cells that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 1.0 mM when the OD₅₅₀ reached avalue of 30. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 62A. The mevalonic acid titer increased over the course of thefermentation to a final value of 23.7 g/L (FIG. 62B). The specificproductivity profile throughout the fermentation is shown in FIG. 62Cand a comparison to FIG. 62A illustrates the de-coupling of growth andmevalonic acid production. The total amount of mevalonic acid producedduring the 51.2 hour fermentation was 140 g from 1.1 kg of utilizedglucose. The molar yield of utilized carbon that went into producingmevalonic acid during fermentation was 15.2%.

V. Isoprene Production from E. coli BL21 (DE3) Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 25 μM when the OD₅₅₀ reached avalue of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀reached 190. The IPTG concentration was raised to 100 μM at 38 hours offermentation. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 63A. The isoprene titer increased over the course of thefermentation to a final value of 2.2 g/L broth (FIG. 63B). The specificproductivity profile throughout the fermentation is shown in FIG. 63Cand a comparison to FIG. 63A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the54.4 hour fermentation was 15.9 g from 2.3 kg of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 1.53%.

VI. Isoprene Production from E. coli BL21 (DE3) Tuner Cells Expressingthe pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) tuner cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 26 μM when the OD₅₅₀ reached avalue of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀reached 175. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 64A. The isoprene titer increased over the course of thefermentation to a final value of 1.3 g/L broth (FIG. 64B). The specificproductivity profile throughout the fermentation is shown in FIG. 64Cand a comparison to FIG. 64A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the48.6 hour fermentation was 9.9 g from 1.6 kg of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 1.34%.

VII. Isoprene Production from E. coli MG1655 Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmidsthat were grown on a plate as explained above in Example 11, part I wereinoculated into a flask containing 500 mL of tryptone-yeast extractmedium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material wasseeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTGconcentration was brought to 24 μM when the OD₅₅₀ reached a value of 45.The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 65A.The isoprene titer increased over the course of the fermentation to afinal value of 393 mg/L broth (FIG. 65B). The specific productivityprofile throughout the fermentation is shown in FIG. 65C and acomparison to FIG. 65A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the67.4 hour fermentation was 2.2 g from 520 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.92%.

VIII. Isoprene Production from E. coli MG1655ack-pta Cells Expressingthe pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655ack-pta cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 30 μM when the OD₅₅₀ reached avalue of 10. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 66A. The isoprene titer increased over the course of thefermentation to a final value of 368 mg/L broth (FIG. 66B). The specificproductivity profile throughout the fermentation is shown in FIG. 66Cand a comparison to FIG. 66A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the56.7 hour fermentation was 1.8 g from 531 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.73%.

IX. Isoprene Production from E. coli FM5 Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

FM5 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmidsthat were grown on a plate as explained above in Example 11, part I wereinoculated into a flask containing 500 mL of tryptone-yeast extractmedium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material wasseeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTGconcentration was brought to 27 μM when the OD₅₅₀ reached a value of 15.The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 67A.The isoprene titer increased over the course of the fermentation to afinal value of 235 mg/L broth (FIG. 67B). The specific productivityprofile throughout the fermentation is shown in FIG. 67C and acomparison to FIG. 67A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the52.3 hour fermentation was 1.4 g from 948 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.32%.

Example 12 Production of Isoprene During the Exponential Growth Phase ofE. coli Expressing Genes from the Mevalonic Acid Pathway and Fermentedin a Fed-Batch Culture

Example 12 illustrates the production of isoprene during the exponentialgrowth phase of cells.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl2*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with ATCC11303 E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-Lbioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 50 hour fermentation was 2.0 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 50 μM when OD₅₅₀ reached 190. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 99. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 1.4 g/L (FIG. 100). The total amount ofisoprene produced during the 50 hour fermentation was 10.0 g. Theprofile of the isoprene specific productivity over time within thebioreactor is shown in FIG. 101. The molar yield of utilized carbon thatcontributed to producing isoprene during fermentation was 1.1%. Theweight percent yield of isoprene from glucose was 0.5%.

Example 13 Flammability Modeling and Testing of Isoprene

I. Summary of Flammability Modeling and Testing of Isoprene

Flammability modeling and experiments were performed for varioushydrocarbon/oxygen/nitrogen/water/carbon dioxide mixtures. This modelingand experimental tested was aimed at defining isoprene andoxygen/nitrogen flammability curves under specified steam and carbonmonoxide concentrations at a fixed pressure and temperature. A matrix ofthe model conditions is shown in Table 9, and a matrix of theexperiments performed is shown in Table 10.

TABLE 9 Summary of Modeled Isoprene Flammability Carbon Steam DioxideIsoprene Oxygen Temperature Pressure Concentration ConcentrationConcentration Concentration Series (° C.) (psig) (wt %) (wt. %) (vol. %)(vol. %) A 40 0 0 0 Varying Varying B 40 0 4 0 Varying Varying C 40 0 05 Varying Varying D 40 0 0 10 Varying Varying E 40 0 0 15 VaryingVarying F 40 0 0 20 Varying Varying G 40 0 0 30 Varying Varying

TABLE 10 Summary of Isoprene Flammability Tests Steam Isoprene OxygenTemperature Pressure Concentration Concentration Concentration SeriesNumber (° C.) (psig) (vol. %) (vol. %) (vol. %) 1 40 0 0 Varying Varying2 40 0 4 Varying VaryingII. Description of Calculated Adiabatic Flame Temperature (CAFT) Model

Calculated adiabatic flame temperatures (CAFT) along with a selectedlimit flame temperature for combustion propagation were used todetermine the flammability envelope for isoprene. The computer programused in this study to calculate the flame temperatures is the NASA GlennResearch Center CEA (Chemical Equilibrium with Applications) software.

There are five steps involved in determining the flammability envelopeusing an adiabatic flame temperature model for a homogeneous combustionmechanism (where both the fuel and oxidant are in the gaseous state):selection of the desired reactants, selection of the test condition,selection of the limit flame temperature, modification of the reactants,and construction of a flammability envelope from calculations.

In this first step, selection of desired reactants, a decision must bemade as to the reactant species that will be present in the system andthe quantities of each. In many cases the computer programs used for thecalculations have a list of reactant and product species. If any of thedata for the species to be studied are not found in the program, theymay be obtained from other sources such as the JANAF tables or from theinternet. In this current model data for water, nitrogen, oxygen andcarbon dioxide were present in the program database. The programdatabase did not have isoprene as a species; therefore the thermodynamicproperties were incorporated manually.

The next step is to decide whether the initial pressure and temperatureconditions that the combustion process is taking place in. In this modelthe pressure was 1 atmosphere (absolute) and the temperature was 40° C.,the boiling point of isoprene.

The limit flame temperature for combustion can be either selected basedon theoretical principles or determined experimentally. Each method hasits own limitations.

Based on prior studies, the limit flame temperatures of hydrocarbonsfall in the range of 1000 K to 1500 K. For this model, the value of 1500K was selected. This is the temperature at which the reaction of carbonmonoxide to carbon dioxide (a highly exothermic reaction and constitutesa significant proportion of the flame energy) becomes self sustaining.

Once the limit flame temperature has been decided upon, modelcalculations are performed on the given reactant mixture (speciesconcentrations) and the adiabatic flame temperature is determined. Flamepropagation is considered to have occurred only if the temperature isgreater than the limit flame temperature. The reactant mixturecomposition is then modified to create data sets for propagation andnon-propagation mixtures.

This type of model shows good agreement with the experimentallydetermined flammability limits. Regions outside the derived envelope arenonflammable and regions within it are flammable. The shape of theenvelope forms a nose. The nose of the envelope is related to thelimiting oxygen concentration (LOC) for gaseous fuels.

III. Results from Calculated Adiabatic Flame Temperature (CAFT) Model

Plotted in FIGS. 68 through 74 are the CAFT model results for Series Ato G, respectively. The figures plot the calculated adiabatic flametemperature (using the NASA CEA program) as a function of fuelconcentration (by weight) for several oxygen/nitrogen ratios (byweight). The parts of the curve that are above 1500 K, the selectedlimit flame temperature, contain fuel levels sufficient for flamepropagation. The results may be difficult to interpret in the formpresented in FIGS. 68 through 74. Additionally, the current form is notconducive to comparison with experimental data which is generallypresented in terms of volume percent.

Using Series A as an example the data in FIG. 68 can be plotted in theform of a traditional flammability envelope. Using FIG. 68 and readingacross the 1500 K temperature line on the ordinate one can determine thefuel concentration for this limit flame temperature by dropping atangent to the abscissa for each curve (oxygen to nitrogen ratio) thatit intersects. These values can then be tabulated as weight percent offuel for a given weight percent of oxidizer (FIG. 75A). Then knowing thecomposition of the fuel (100 wt. % isoprene) and the composition of theoxidizer (relative content of water, oxygen and nitrogen) molarquantities can be established.

From these molar quantities percentage volume concentrations can becalculated. The concentrations in terms of volume percent can then beplotted to generate a flammability envelope (FIG. 75B). The area boundedby the envelope is the explosible range and the area excluded is thenon-explosible range. The “nose” of the envelope is the limiting oxygenconcentration. FIGS. 76A and 76B contain the calculated volumeconcentrations for the flammability envelope for Series B generated fromdata presented in FIG. 69. A similar approach can be used on datapresented in FIGS. 70-74.

IV. Flammability Testing Experimental Equipment and Procedure

Flammability testing was conducted in a 4 liter high pressure vessel.The vessel was cylindrical in shape with an inner diameter of 6″ and aninternal height of 8.625″. The temperature of the vessel (and the gasesinside) was maintained using external heaters that were controlled by aPID controller. To prevent heat losses, ceramic wool and reflectiveinsulation were wrapped around the pressure vessel. Type K thermocoupleswere used the measure the temperature of the gas space as well as thetemperature of the vessel itself. FIG. 77 illustrates the test vessel.

Before a test was ran, the vessel was evacuated and purged with nitrogento ensure that any gases from previous tests were removed. A vacuum wasthen pulled on the vessel. The pressure after this had been done wastypically around 0.06 bar(a). Due to the nitrogen purging, the gasresponsible for this initial pressure was assumed to be nitrogen. Usingpartial pressures, water, isoprene, nitrogen, and oxygen were then addedin the appropriate amounts to achieve the test conditions in question. Amagnetically driven mixing fan within the vessel ensured mixing of thegaseous contents. The gases were allowed to mix for about 2 minutes withthe fan being turned off approximately 1 minute prior to ignition.

The igniter was comprised of a 1.5 ohm nicrome coil and an AC voltagesource on a timer circuit. Using an oscilloscope, it was determined that34.4 VAC were delivered to the igniter for 3.2 seconds. A maximumcurrent of 3.8 amps occurred approximately halfway into the ignitioncycle. Thus, the maximum power was 131 W and the total energy providedover the ignition cycle was approximately 210 J.

Deflagration data was acquired using a variable reluctance ValidyneDP215 pressure transducer connected to a data acquisition system. A gasmixture was considered to have deflagrated if the pressure rise wasgreater than or equal to 5%.

V. Results of Flammability Testing

The first experimental series (Series 1) was run at 40° C. and 0 psigwith no steam. Running tests at varying concentrations of isoprene andoxygen produced the flammability curve shown in FIG. 78A. The datapoints shown in this curve are only those that border the curve. Adetailed list of all the data points taken for this series is shown inFIGS. 80A and 80B.

FIG. 78B summarizes the explosibility data points shown in FIG. 78A.FIG. 78C is a comparison of the experimental data with the CAFT modelpredicted flammability envelope. The model agrees very well with theexperimental data. Discrepancies may be due to the non-adiabatic natureof the test chamber and limitations of the model. The model looks at aninfinite time horizon for the oxidation reaction and does not take intoconsideration any reaction kinetic limitation.

Additionally, the model is limited by the number of equilibrium chemicalspecies that are in its database and thus may not properly predictpyrolytic species. Also, the flammability envelope developed by themodel uses one value for a limit flame temperature (1500K). The limitflame temperature can be a range of values from 1,000K to 1,500Kdepending on the reacting chemical species. The complex nature ofpyrolytic chemical species formed at fuel concentrations above thestoichiometric fuel/oxidizer level is one reason why the model may notaccurately predict the upper flammable limit for this system.

The second experimental series (Series 2) was run at 40° C. and 0 psigwith a fixed steam concentration of 4%. Running tests at varyingconcentrations of isoprene and oxygen produced the flammability curveshown in FIG. 79A. The data points shown in this curve are only thosethat border the curve. A detailed list of all the data points taken forthis series is shown in FIG. 81. Due to the similarity between the datain Series 1 only the key points of lower flammable limit, limitingoxygen concentration, and upper flammable limits were tested. Theaddition of 4% steam to the test mixture did not significantly changethe key limits of the flammability envelope. It should be noted thathigher concentrations of steam/water and or other inertants mayinfluence the flammability envelope.

FIG. 79B summarizes the explosibility data points shown in FIG. 79A.FIG. 79C is a comparison of the experimental data with the CAFT modelpredicted flammability envelope. The model agrees very well with theexperimental data. Discrepancies may be due to the same factorsdescribed in Series 1

V. Calculation of Flammability Limits of Isoprene in Air at 3Atmospheres of Pressure

The methods described in Example 13, parts I to IV were also used tocalculate the flammability limits of isoprene at an absolute systempressure of 3 atmospheres and 40° C. These results were compared tothose of Example 13, parts Ito IV at an absolute system pressure of 1atmosphere and 40° C. This higher pressure was tested because theflammability envelope expands or grows larger as the initial systempressure is increased. The upper flammability limit is affected themost, followed by the limiting oxygen composition. The lowerflammability limit is the least affected (see, for example, “Bulletin627—Flammability Characteristics of Combustible Gases and Vapors”written by Michael G. Zabetakis and published by the former US Bureau ofMines (1965), which is hereby incorporated by reference in its entirety,particular with respect to the calculation of flammability limits).

In FIG. 82, the calculated adiabatic flame temperature is plotted as afunction of isoprene (fuel) concentration, expressed in weight percentof the total fuel/nitrogen/oxygen, where the system pressure wasinitially 3 atmospheres. The calculated flame temperatures are verysimilar to those determined initially in the 1 atmosphere system (FIG.83). As a result, when flammability envelopes are generated using thecalculated adiabatic flammability data, the curves are very similar (seeFIGS. 84 and 85). Therefore, based on these theoretical calculations, asystem pressure increase from 1 atmosphere to 3 atmosphere does notresult in a significant increase/broadening of the flammabilityenvelope. If desired, these model results may be validated usingexperimental testing (such as the experimental testing described hereinat a pressure of 1 atmosphere).

VII. Summary of Flammability Studies

A calculated adiabatic temperature model was developed for theflammability envelope of the isoprene/oxygen/nitrogen/water/carbondioxide system at 40° C. and 0 psig. The CAFT model that was developedagreed well with the experimental data generated by the tests conductedin this work. The experimental results from Series 1 and 2 validated themodel results from Series A and B.

Example 14 Production of Isoprene in E. coli Expressing M. mazeiMevalonate Kinase and P. alba Isoprene Synthase

I. Construction of Vectors and Strains Encoding M. mazei MevalonateKinase (MVK) and P. alba Isoprene Synthase

(i) Construction of strain EWL201 (BL21, Cm-GI1.2-KKDyI)

E. coli BL21 (Novagen brand, EMD Biosciences, Inc.) was a recipientstrain, transduced with MCM331 P1 lysate (lysate prepared according tothe method described in Ausubel, et al., Current Protocols in MolecularBiology. John Wiley and Sons, Inc.). Transductants were selected for byspreading cells onto L Agar and 20 chloramphenicol. The plates wereincubated overnight at 30° C. Analysis of transductants showed nocolonies on control plates (water+cells control plate for reversion andwater and P1 lysate control plate for lysate contamination.

Four transductants were picked and used to inoculate 5 mL L Broth and 20μg/μl chloramphenicol. The cultures were grown overnight at 30° C. withshaking at 200 rpm. To make genomic DNA preps of each transductant forPCR analysis, 1.5 mL of overnight cell culture were centrifuged. Thecell pellet was resuspended with 400 μl Resuspension Buffer (20 mM Tris,1 mM EDTA, 50 mM NaCl, pH 7.5) and 4 μl RNase, DNase-free (Roche) wasadded. The tubes were incubated at 37° C. for 30 minutes followed by theaddition of 4 μl 10% SDS and 4 μl of 10 mg/ml Proteinase K stocksolution (Sigma-Aldrich). The tubes were incubated at 37° C. for 1 hour.The cell lysate was transferred into 2 ml Phase Lock Light Gel tubes(Eppendorf) and 200 μl each of saturated phenol pH7.9 (Ambion Inc.) andchloroform were added. The tubes were mixed well and microcentrifugedfor 5 minutes. A second extraction was done with 400 μl chloroform andthe aqueous layer was transferred to a new eppendorf tube. The genomicDNA was precipitated by the addition of 1 ml of 100% ethanol andcentrifugation for 5 minutes. The genomic DNA pellet was washed with 1ml 70% ethanol. The ethanol was removed and the genomic DNA pellet wasallowed to air dry briefly. The genomic DNA pellet was resuspended with200 μl TE.

Using Pfu Ultra II DNA polymerase (Stratagene) and 200 ng/μl of genomicDNA as template, 2 different sets of PCR reaction tubes were preparedaccording to manufacturer's protocol. For set 1, primers MCM130 and GBCm-Rev (Table 11) were used to ensure transductants were successfullyintegrated into the attTn7 locus. PCR parameters for set 1 were 95° C.for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25seconds, 72° C. for 25 seconds (repeat steps 2-4 for 28 cycles), 72° C.for 1 minute. For set 2, primers MVD For and MVD Rev (Table 11) wereused to ensure that the gi1.2-KKDyI operon integrated properly. PCRparameters for set 2 were 95° C. for 2 minutes (first cycle only), 95°C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 10 seconds (repeatsteps 2-4 for 28 cycles), 72° C. for 1 minute. Analysis of PCR ampliconson a 1.2% E-gel (Invitrogen Corp.) showed that all 4 transductant cloneswere correct (picked one and designated as strain EWL201).

ii) Construction of Strain EWL204 (BL21, Loopout-GI1.2-KKDyI)

The chloramphenicol marker was looped out of strain EWL201 using plasmidpCP20 as described by Datsenko and Wanner (2000) (Datsenko et al., ProcNatl. Acad. Sci. USA 97:6640-6645, 2000). One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products. (Datsenkoet al., PNAS, 97: 6640-6645, 2000). EWL201 cells were grown in L Brothto midlog phase and then washed three times in ice-cold, sterile water.An aliquot of 50 μl of cell suspension was mixed with 1 μl of pCP20 andthe cell suspension mixture was electroporated in a 2 mm cuvette(Invitrogen Corp.) at 2.5 Volts and 25 uFd using a Gene PulserElectroporator (Bio-Rad Inc.). 1 ml of LB was immediately added to thecells, then transferred to a 14 ml polypropylene tube (Sarstedt) with ametal cap.

Cells were allowed to recover by growing for 1 hour at 30° C.Transformants were selected on L Agar and 20 μg/μl chloramphenicol and50 μg/μl carbenicillin and incubated at 30° C. overnight. The next day,a single clone was grown in 10 ml L Broth and 50 μg/μl carbenicillin at30° C. until early log phase. The temperature of the growing culture wasthen shifted to 42° C. for 2 hours. Serial dilutions were made, thecells were then spread onto LA plates (no antibiotic selection), andincubated overnight at 30° C. The next day, 20 colonies were picked andpatched onto L Agar (no antibiotics) and LA and 20 μg/μl chloramphenicolplates. Plates were then incubated overnight at 30° C. Cells able togrow on LA plates, but not LA and 20 μg/μl chloramphenicol plates, weredeemed to have the chloramphenicol marker looped out (picked one anddesignated as strain EWL204).

iii) Construction of Plasmid pEWL230 (pTrc P. alba)

Generation of a synthetic gene encoding Populus alba isoprene synthase(P. alba HGS) was outsourced to DNA2.0 Inc. (Menlo Park, Calif.) basedon their codon optimization method for E. coli expression. The syntheticgene was custom cloned into plasmid pET24a (Novagen brand, EMDBiosciences, Inc.) and delivered lyophilized (FIGS. 112, 113A and 113B).

A PCR reaction was performed to amplify the P. alba isoprene synthase(P. alba HGS) gene using pET24 P. alba HGS as the template, primersMCM182 and MCM192, and Herculase II Fusion DNA polymerase (Stratagene)according to manufacturer's protocol. PCR conditions were as follows:95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C.for 20 seconds, 72° C. for 1 minute, repeat for 25 cycles, with finalextension at 72° C. for 3 minutes. The P. alba isoprene synthase PCRproduct was purified using QIAquick PCR Purification Kit (Qiagen Inc.).

P. alba isoprene synthase PCR product was then digested in a 20 μlreaction containing 1 μl BspHI endonuclease (New England Biolabs) with 2μl 10×NEB Buffer 4. The reaction was incubated for 2 hours at 37° C. Thedigested PCR fragment was then purified using the QIAquick PCRPurification Kit. A secondary restriction digest was performed in a 20μl reaction containing 1 μl PstI endonuclease (Roche) with 2 μl 10×Buffer H. The reaction was incubated for 2 hours at 37° C. The digestedPCR fragment was then purified using the QIAquick PCR Purification Kit.Plasmid pTrcHis2B (Invitrogen Corp.) was digested in a 20 μl reactioncontaining 1 μl NcoI endonuclease (Roche), 1 μl PstI endonuclease, and 2μl 10× Buffer H. The reaction was incubated for 2 hours at 37° C. Thedigested pTrcHis2B vector was gel purified using a 1.2% E-gel(Invitrogen Corp.) and extracted using the QIAquick Gel Extraction Kit(Qiagen) (FIG. 114). Using the compatible cohesive ends of BspHI andNcoI sites, a 20 μl ligation reaction was prepared containing 5 μl P.alba isoprene synthase insert, 2 μl pTrc vector, 1 μl T4 DNA ligase (NewEngland Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH₂O. The ligationmixture was incubated at room temperature for 40 minutes. The ligationmixture was desalted by floating a 0.025 μm nitrocellulose membranefilter (Millipore) in a petri dish of ddH₂O and applying the ligationmixture gently on top of the nitrocellulose membrane filter for 30minutes at room temperature. MCM446 cells (See section II) were grown inLB to midlog phase and then washed three times in ice-cold, sterilewater. An aliquot of 50 μl of cell suspension was mixed with 5 μl ofdesalted pTrc P. alba HGS ligation mix. The cell suspension mixture waselectroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a GenePulser Electroporator. 1 ml of LB is immediately added to the cells,then transferred to a 14 ml polypropylene tube (Sarstedt) with a metalcap.

Cells were allowed to recover by growing for 2 hour at 30° C.Transformants were selected on L Agar and 50 μg/μl carbenicillin and 10mM mevalonic acid and incubated at 30° C. The next day, 6 transformantswere picked and grown in 5 ml L Broth and 50 μg/μl carbenicillin tubesovernight at 30° C. Plasmid preps were performed on the overnightcultures using QIAquick Spin Miniprep Kit (Qiagen). Due to the use ofBL21 cells for propagating plasmids, a modification of washing the spincolumns with PB Buffer 5× and PE Buffer 3× was incorporated to thestandard manufacturer's protocol for achieving high quality plasmid DNA.Plasmids were digested with PstI in a 20 μl reaction to ensure thecorrect sized linear fragment. All 6 plasmids were the correct size andshipped to Quintara Biosciences (Berkeley, Calif.) for sequencing withprimers MCM65, MCM66, EL1000 (Table 11). DNA sequencing results showedall 6 plasmids were correct. Picked one and designated plasmid as EWL230(FIGS. 57, 58A and 58B).

iv) Construction of Plasmid pEWL244 (pTrc P. alba-mMVK)

A PCR reaction was performed to amplify the Methanosarcina mazei (M.mazei) MVK gene using MCM376 as the template (see section v), primersMCM165 and MCM177 (see Table 11), and Pfu Ultra II Fusion DNA polymerase(Stratagene) according to manufacturer's protocol. PCR conditions wereas follows: 95° C. for 2 minutes (first cycle only), 95° C. for 25seconds, 55° C. for 25 seconds, 72° C. for 18 seconds, repeat for 28cycles, with final extension at 72° C. for 1 minute. The M. mazei MVKPCR product was purified using QIAquick PCR Purification Kit (QiagenInc.).

The M. mazei MVK PCR product was then digested in a 40 μl reactioncontaining 8 μl PCR product, 2 μl PmeI endonuclease (New EnglandBiolabs), 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 22 μl of ddH₂O. Thereaction was incubated for 3 hours at 37° C. The digested PCR fragmentwas then purified using the QIAquick PCR Purification Kit. A secondaryrestriction digest was performed in a 47 μl reaction containing 2 μlNsiI endonuclease (Roche), 4.7 μl 10× Buffer H, and 40 μl of PmeIdigested M. mazei MVK fragment. The reaction was incubated for 3 hoursat 37° C. The digested PCR fragment was then gel purified using a 1.2%E-gel and extracted using the QIAquick Gel Extraction Kit. PlasmidEWL230 was digested in a 40 μl reaction containing 10 μl plasmid, 2 μlPmeI endonuclease, 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 20 μl ofddH₂O. The reaction was incubated for 3 hours at 37° C. The digested PCRfragment was then purified using the QIAquick PCR Purification Kit. Asecondary restriction digest was performed in a 47 μl reactioncontaining 2 μl PstI endonuclease, 4.7 μl 10× Buffer H, and 40 μl ofPmeI digested EWL230 linear fragment. The reaction was incubated for 3hours at 37° C. The digested PCR fragment was then gel purified using a1.2% E-gel and extracted using the QIAquick Gel Extraction Kit (FIG.117). Using the compatible cohesive ends of NsiI and PstI sites, a 20 μlligation reaction was prepared containing 8 μl M. mazei MVK insert, 3 μlEWL230 plasmid, 1 μl T4 DNA ligase, 2 μl 10× ligase buffer, and 6 μlddH₂O. The ligation mixture was incubated at overnight at 16° C. Thenext day, the ligation mixture was desalted by floating a 0.025 μmnitrocellulose membrane filter in a petri dish of ddH₂O and applying theligation mixture gently on top of the nitrocellulose membrane filter for30 minutes at room temperature. MCM446 cells were grown in LB to midlogphase and then washed three times in ice-cold, sterile water. An aliquotof 50 μl of cell suspension was mixed with 5 μl of desalted pTrc P.alba-mMVK ligation mix. The cell suspension mixture was electroporatedin a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene PulserElectroporator. 1 ml of LB is immediately added to the cells, then thecells are transferred to a 14 ml polypropylene tube with a metal cap.Cells were allowed to recover by growing for 2 hour at 30° C.Transformants were selected on LA and 50 μg/μl carbenicillin and 5 mMmevalonic acid plates and incubated at 30° C. The next day, 6transformants were picked and grown in 5 ml LB and 50 μg/μlcarbenicillin tubes overnight at 30° C. Plasmid preps were performed onthe overnight cultures using QIAquick Spin Miniprep Kit. Due to the useof BL21 cells for propagating plasmids, a modification of washing thespin columns with PB Buffer 5× and PE Buffer 3× was incorporated to thestandard manufacturer's protocol for achieving high quality plasmid DNA.Plasmids were digested with PstI in a 20 μl reaction to ensure thecorrect sized linear fragment. Three of the 6 plasmids were the correctsize and shipped to Quintara Biosciences for sequencing with primersMCM65, MCM66, EL1000, EL1003, and EL1006 (Table 11). DNA sequencingresults showed all 3 plasmids were correct. Picked one and designatedplasmid as EWL244 (FIGS. 118, 119A and 199B).

v) Construction of Plasmid MCM376-MVK from M. mazei Archaeal Lower inpET200D.

The MVK ORF from the M. mazei archaeal Lower Pathway operon (FIGS.131A-C) was PCR amplified using primers MCM161 and MCM162 (Table 11)using the Invitrogen Platinum HiFi PCR mix. 45 μL of PCR mix wascombined with 1 μL template, 1 μL of each primer at 10 μM, and 2 μLwater. The reaction was cycled as follows: 94° C. for 2:00 minutes; 30cycles of 94° C. for 0:30 minutes, 55° C. for 0:30 minutes and 68° C.for 1:15 minutes; and then 72° C. for 7:00 minutes, and 4° C. untilcool. 3 μL of this PCR reaction was ligated to Invitrogen pET200Dplasmid according to the manufacturer's protocol. 3 μL of this ligationwas introduced into Invitrogen TOP10 cells, and transformants wereselected on LA/kan50. A plasmid from a transformant was isolated and theinsert sequenced, resulting in MCM376 (FIGS. 131A-C).

vi) Construction of Strain EWL251 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P.alba-mMVK)

MCM331 cells (which contain chromosomal construct gi1.2KKDyI encoding S.cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonatepyrophosphate decarboxylase, and IPP isomerase) were grown in LB tomidlog phase and then washed three times in ice-cold, sterile water.Mixed 50 μl of cell suspension with 1 μl of plasmid EWL244. The cellsuspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediatelyadded to the cells, then the cells were transferred to a 14 mlpolypropylene tube with a metal cap. Cells were allowed to recover bygrowing for 2 hours at 30° C. Transformants were selected on LA and 50μg/μl carbenicillin and 5 mM mevalonic acid plates and incubated at 37°C. One colony was selected and designated as strain EWL251.

vii) Construction of Strain EWL256 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P.alba-mMVK, pCL Upper MVA)

EWL251 cells were grown in LB to midlog phase and then washed threetimes in ice-cold, sterile water. Mixed 50 μl of cell suspension with 1μl of plasmid MCM82 (which is pCL PtrcUpperPathway encoding E. faecalismvaE and mvaS). The cell suspension mixture was electroporated in a 2 mmcuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 mlof LB was immediately added to the cells, then transferred to a 14 mlpolypropylene tube with a metal cap. Cells were allowed to recover bygrowing for 2 hour at 30° C. Transformants were selected on LA and 50μg/μl carbenicillin and 50 μg/μl spectinomycin plates and incubated at37° C. Picked one colony and designated as strain EWL256.

TABLE 11 Primer Sequences Primer name Primer sequence MCM130ACCAATTGCACCCGGCAGA (SEQ ID NO: 109) GB CmGCTAAAGCGCATGCTCCAGAC (SEQ ID NO: 110) Rev MVDGACTGGCCTCAGATGAAAGC (SEQ ID NO: 111) For MVDCAAACATGTGGCATGGAAAG (SEQ ID NO: 112) Rev MCM182GGGCCCGTTTAAACTTTAACTAGACTCTGCAGTTAGCGTTCAAA CGGCAGAA (SEQ ID NO: 113)MCM192 CGCATGCATGTCATGAGATGTAGCGTGTCCACCGAAAA (SEQ ID NO: 114) MCM65ACAATTTCACACAGGAAACAGC (SEQ ID NO: 115) MCM66CCAGGCAAATTCTGTTTTATCAG (SEQ ID NO: 89) EL1000GCACTGTCTTTCCGTCTGCTGC (SEQ ID NO: 117) MCM165GCGAACGATGCATAAAGGAGGTAAAAAAACATGGTATCCTGTTCTGCGCCGGGTAAGATTTACCTG (SEQ ID NO: 118) MCM177GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCAGAC CTTGC (SEQ ID NO: 119)EL1003 GATAGTAACGGCTGCGCTGCTACC (SEQ ID NO: 120) EL1006GACAGCTTATCATCGACTGCACG (SEQ ID NO: 121) MCM161CACCATGGTATCCTGTTCTGCG (SEQ ID NO: 122) MCM162TTAATCTACTTTCAGACCTTGC (SEQ ID NO: 123)II. Construction of MCM442-449: BL21 and BL21(DE3) withFRT-cmR-FRT-gi1.x-mKKDyIi) Construction of Template for Recombination

FRT-based recombination cassettes, and plasmids for Red/ET-mediatedintegration and antibiotic marker loopout were obtained from GeneBridges GmbH (Germany). Procedures using these materials were carriedout according to Gene Bridges protocols. Primers MCM193 and MCM195 wereused to amplify the resistance cassette from the FRT-gb2-Cm-FRT templateusing Stratagene Herculase II Fusion kit according to the manufacturer'sprotocol. The 50 μL reaction was cycled as follows: 95° C., 2 minutes;(95° C., 20 seconds, 55° C., 20 seconds, 72° C., 1 minute)×5, (95° C.,20 seconds, 60° C., 20 seconds, 72° C., 1 minute)×25; 72° C., 3 minutes;4° C. until cool. The amplicon was purified by a Qiagen PCR columnaccording to the manufacturer's protocol and eluted in 30 μL EB (ElutionBuffer). DNA was digested with NdeI and PciI in a 20 μL reaction with 1×Roche H buffer and 0.5 μL BSA. Plasmid MCM376 was digested in a 10 μLreaction containing 1 μL each of NdeI, NcoI, and Roche H buffer.Reactions proceeded overnight at 37° C., and then cut DNA was purifiedon Qiagen PCR columns and eluted in 30 μL EB. The PCR product wasligated into MCM376 in a reaction containing 1 μL vector, 3 μL PCRproduct, 1 μL Roche Quick Ligase Buffer 2, 5 μL Buffer1, and 1 μLLigase. The reaction proceeded at room temperature for 3 hours and then5 μL was transformed into Invitrogen TOP10 cells according to themanufacturer's protocol. Transformants were selected on L agar (LA) andchloramphenicol (10 μg/mLO) at 37° C. overnight.

Transformant colonies were patched onto LA containing chloramphenicol(30 μg/mL) and kanamycin (50 μg/ml) for storage and sent to Quintara(Berkeley, Calif.) for sequencing. Four clones, one each with the fourdifferent nucleotides at the “N” in primer MCM195, were found to havethe correct sequence for the inserted promoter. Clones were grown in 5mL LB containing chloramphenicol (30 μg/mL) and kanamycin (50 μg/mL) andused for the preparation of plasmid DNA. This plasmid was retransformedinto TOP10 cells and strains were frozen as:

TABLE 12 MCM484-487 MCM484 cmR-gi1.6-MVK(mazei) in pET (clone A1-3,variable nt A) MCM485 cmR-gi1.0-MVK(mazei) in pET (clone B4-6, variablent C) MCM486 cmR-gi1.2-MVK(mazei) in pET (clone C1-5, variable nt G)MCM487 cmR-gi1.5-MVK(mazei) in pET (clone C3-3, variable nt T)ii) Creation of Recombination Target Strains MCM349 and MCM441

The chloramphenicol resistance (cmR) marker was looped out of strainMCM331 using plasmid pGB706 (GeneBridges) according to Manufacturer'sinstructions. MCM331 cells were grown to mid-log in LB and washed threetimes in iced, sterile water. A 1 μL aliquot of pGB706 DNA was added to50 μL of cell suspension and this mixture was electroporated in a 2 mmcuvette at 2.5 volts, 25 uFd followed immediately by recovery in 500 μLLB for one hour at 30 C. Transformants were selected on LB containingtetracycline (5 μg/ml) at 30° C. The following day, a clone was grown upat 30° C. in LB containing tetracycline (5 μg/ml) until visibly turbid(OD₆₀₀˜0.5-0.8). This culture was streaked onto LB and grown overnightat 37° C. A clone that was unable to grow on LB containingchloramphenicol (10 μg/mL) or LB containing tetracycline (5 μg/mL) wasfrozen as MCM348. Plasmid MCM356 (pRedET carbencillin; GeneBridges) waselectroporated in as described above and transformants were selected onLB containing carbenicillin (50 μg/mL) at 30° C. A clone was grown in LBcarbenicillin (50 μg/mL) at 30° C. and frozen as MCM349.

Strain MCM441 was Created by Electrotransforming Plasmid MCM356 intoEWL204 as Above.

iii) Recombination of FRT-cmR-FRT-gi1.x-mMVK into MCM349 and MCM441

Plasmids MCM484-487 were used as template for PCR amplification withprimers MCM120 and MCM196 and Herculase II Fusion kit, according to themanufacturer's protocol. Three reactions per template were carried out,with 0, 1, or 3 μL DMSO. The 50 μL reactions were cycled as follows: 95°C., 2 minutes; (95° C., 20 seconds; 55° C. 20 seconds; 72° C., 1.5minutes) for five cycles; (95° C., 20 seconds; 60° C. 20 seconds; 72°C., 1.5 minutes) for 25 cycles; 72° C. for 3 minutes; 4° C., overnight.The three reactions from a given template were pooled and purified onQiagen PCR columns and eluted with 30 μL EB at 60° C. 5 μL DNA wasdigested with 1 μL DpnI in 1× Roche Buffer A for 3 hours at 37° C. ThisDNA was then microdialyzed against excess water for 30 minutes.

Strains were grown in 5 mL LB containing carbenicillin (50 μg/mL) fromfresh streaks at 30 C to an OD600 of ˜0.5. 40 mM L-arabinose was addedand cultures were incubated at 37° C. for 1.5 hours. Cells wereharvested and electroporated with 3 μL dialyzed amplicons above, andthen recovered in 500 μL SOC at 37 C for 1.5-3 hours. Transformants wereselected on LA plates containing chloramphenicol (5 μg/mL) at 37° C.

Kanamycin sensitive clones were screened by PCR for insertion of theamplicon. PCR products from positive clones were sequenced to verify thesequence of inserted DNA. Amplicons were consistent with theFRT-gi1.2-yKKDyI at attTn7 in MCM441 and 348 being replaced byFRT-cmR-FRT-gi1.x-mKKDyI (The yK and mK designations refer to themevalonate kinase from Saccharomyces cerevisiae and Methanosarcina mazeirespectively).

TABLE 13A The following strains were grown in LB containingchloramphenicol (5 μg/mL) and frozen. Recom- bination Amplicon Strain IDName Parent Template MCM442 BL21(DE3) cmR-gi1.6mKKDyI A1, MCM349 MCM484clone37 (A) MCM443 BL21(DE3) cmR-gi1.0mKKDyI B4, MCM349 MCM485 clone27(C) MCM444 BL21(DE3) cmR-gi1.2mKKDyI C1, MCM349 MCM486 clone16 (G)MCM445 BL21(DE3) cmR-gi1.5mKKDyI MCM349 MCM487 C3, clone7 (T) MCM446BL21 cmR-gi1.6mKKDyI A1-3 (A) MCM441 MCM484 MCM447 BL21 cmR-gi1.0mKKDyIB4-6 (C) MCM441 MCM485 MCM448 BL21 cmR-gi1.2mKKDyI C1-5 (G) MCM441MCM486 MCM449 BL21 cmR-gi1.5mKKDyI C3-3 (T) MCM441 MCM487

TABLE 13B Primers MCM120 AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAGCAATTAACCCTCACTAAAGGGCGG (SEQ ID NO: 97) MCM193GATATACATATGAATTAACCCTCACTAAAGG (SEQ ID NO: 124) MCM195GCATGCATGACATGTTTTTTTACCTCCTTTGTTATCCGCTCACAATTAGTGGTTGAATTATTTGCTCAGGATGTGGCATNGTCAAGGGCGCGGCCGCGATCTAATACGACTCACTATAGGGCTCG (SEQ ID NO: 125) MCM196AGGCTCTCAACTCTGACATGTTTTTTTCCTCCTTAAGGGTGCAGGCCTATCGCAAATTAGCTTAATCTACTTTCAGACCTTGCTCGG (SEQ ID NO: 126)III. The Effect of Yeast Extract on Isoprene Production in E. coliExpressing Genes from the Mevalonic Acid Pathway and Grown in Fed-BatchCulture at the 15-L ScaleMedium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized witha 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCL Upper), theintegrated lower MVA pathway (gi1.2KKDyI), and high expression ofmevalonate kinase from M. mazei and isoprene synthase from P. alba(pTrcAlba-mMVK). This experiment was carried out to monitor isopreneformation from glucose at the desired fermentation pH 7.0 andtemperature 30° C. A frozen vial of the E. coli strain was thawed andinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 15-Lbioreactor bringing the initial volume to 5-L.

i) Production of Isoprene in E. Coli Cells (EL256) Grown in Fed-BatchCulture without Yeast Extract Feeding

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 67 hour fermentation was 3.9 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 102 μM when the optical density at 550nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to192 μM when OD₅₅₀ reached 140. The OD₅₅₀ profile within the bioreactorover time is shown in FIG. 125A. The isoprene level in the off gas fromthe bioreactor was determined using a Hiden mass spectrometer. Theisoprene titer increased over the course of the fermentation to a finalvalue of 35.6 g/L (FIG. 125B). The total amount of isoprene producedduring the 67 hour fermentation was 320.6 g and the time course ofproduction is shown in FIG. 125C. The metabolic activity profile, asmeasured by TCER, is shown in FIG. 125D. The molar yield of utilizedcarbon that went into producing isoprene during fermentation was 17.9%.The weight percent yield of isoprene from glucose was 8.1%.

ii) Production of Isoprene in E. coli cells (EL256) Grown in Fed-BatchCulture with Yeast Extract Feeding

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 68 hour fermentation was 7.1 kg. A total of 1.06kg of yeast extract was also fed during the fermentation. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 208 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 7. TheIPTG concentration was raised to 193 μM when OD₅₅₀ reached 180. TheOD₅₅₀ profile within the bioreactor over time is shown in FIG. 126A. Theisoprene level in the off gas from the bioreactor was determined using aHiden mass spectrometer. The isoprene titer increased over the course ofthe fermentation to a maximum value of 32.2 g/L (FIG. 126B). The totalamount of isoprene produced during the 68 hour fermentation was 395.5 gand the time course of production is shown in FIG. 126C. The time courseof volumetric productivity is shown in FIG. 126D and shows that anaverage rate of 1.1 g/L/hr was maintained for between 23 and 63 hours.The metabolic activity profile, as measured by CER, is shown in FIG.126D. The molar yield of utilized carbon that went into producingisoprene during fermentation was 10.3%. The weight percent yield ofisoprene from glucose was 5.2%.

IV. Production of Isoprene from Different Carbon Sources in E. coliHarboring the Mevalonic Acid (MVA) Pathway and Isoprene Synthase(EWL256)

Media Recipe (Per Liter Fermentation Media):

K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 0.2 g,1000× Modified Trace Metal Solution 1 ml. All of the components weredissolved sequentially in diH₂O. The pH was adjusted to 6.8 withammonium hydroxide (30%) and brought to volume. Media was filtersterilized with a 0.22 micron filter. Carbon source was added to a finalconcentration of 1%. Required antibiotics were added after sterilizationand pH adjustment.

1000× Trace Metal Solution (Per Liter Fermentation Media):

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in diH₂O,pH to 3.0 with HCl/NaOH, and then brought to volume and filtersterilized with a 0.22 micron filter.

i) Preparation of AFEX Biomass Hydrolysate

AFEX pretreated corn stover was hydrolyzed to prepare biomasshydrolysate containing both xylose, glucose and acetate.

AFEX pretreated corn stover, received from Michigan BiotechnologyInstitute, was used. The pretreatment conditions were, 60% moisture, 1:1ammonia loading, and 90° C. for 30 minutes, then air dried. The moisturecontent in the AFEX pretreated corn stover was 21.27%. Content of glucanand xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (drybasis) respectively. The enzyme used was accellerase 1000, GrindamylH121 (Danisco xylanase product from Aspergillus niger for bread-makingindustry).

For saccharification, 20 g of AFEX pretreated corn stover was added intoa 500 ml flask, together with 5 ml of 1 M pH 4.8 sodium citrate buffer,2.25 ml of Accellerase 1000, 0.1 ml of Grindamyl H121, and 72.65 ml ofDI water. The flask was put in an orbital shaker, and incubated at 50°C. for 96 hours.

For analysis, one sample was taken from the shaker, and analyzed usingHPLC. The hydrolysate contained 37.2 g/l of glucose and 24.3 g/L ofxylose, and 7.6 g/L of oligomers of glucose and/or xylose. Additionally,the hydrolysate also contains 1.17 g/L acetate.

ii) Experimental Procedure

An inoculum of the E. coli strain EWL256 containing the MVA pathway andisoprene synthase was taken from a frozen vial and streaked onto an LBbroth agar plate containing spectinomycin (50 μg/mL) and carbinicllin(50 μg/mL) and incubated at 30° C. overnight. A single colony wasinoculated into TM3 media containing glucose, xylose, glycerol, acetateor biomass as only carbon source and grown overnight at 30° C. Cellsgrow on acetate reached a significantly lower optical density. Cellsgrown on glucose, glycerol, biomass hydrolysate or acetate were dilutedinto 20 mL of TM3 media containing the respective carbon sources toreach an optical density of between 0.1 measured at 600 nM. A negativecontrol not containing any carbon source was prepared from the glucoseovernight culture. A separate experiment was performed with glucose andxylose, where the cultures were diluted to an optical density of 0.05.All culture conditions (except for acetate and glycerol) were tested induplicates and the presented results are averaged between thesecultures. Production of isoprene was induced with 200 μM IPTG from thebeginning of the experiment. The flasks were incubated at 30° C. in anorbital shaker (200 rpm) and growth was followed by measuring opticaldensity. After the glucose fed cultures had reached an optical densityof approximately 0.4, samples were analyzed for isoprene production fromall the tested carbon sources every hour for three hours. Samples of 100μL were transferred in duplicates to 2 mL glass vials, sealed andincubated for 30 min at 30° C. The bacteria were then heat killed byincubation at 80° C. for 8 minutes. The amount of produced isoprene wasmeasured using GC-MS and specific productivity (μg/L*hr) was calculated.

iii) Results

Significant production of isoprene could be demonstrated during growthon all the tested carbon sources. These carbon sources are examples ofcommon alcohols, organic acids, sugars containing 5 or 6 carbon units(C₅ or C₆), and biomass hydrolysate.

The initial growth rate on biomass hydrolysate was comparable to thegrowth rate on glucose (FIG. 127A). The initial specific productivityduring growth on biomass hydrolysate was significantly higher thanduring growth on glucose. This demonstrates that biomass hydrolysate canbe used as an efficient source of carbon for the production of isoprene.The specific productivity declined after 255 minutes of growth onbiomass hydrolysate (FIG. 127B). The bacteria had a slower growth ratewith xylose as only carbon source when compared to glucose (FIG. 127C),but a significant specific isoprene productivity was measured (FIG.127D). This shows that both C₅ and C₆ sugars can be utilized for theproduction of isoprene via the mevalonate acid pathway.

Suprisingly, bacteria grown on acetate as the only carbon source had aspecific productivity of isoprene approximately twice as high as duringgrowth on glucose (FIG. 127A). The bacteria grew slower on acetate whencompared to glucose (FIG. 127B), but the performed experimentdemonstrates that acetate can also be used as a carbon source for theproduction of isoprene. Acetate was also present in the biomasshydrolysate as measured by HPLC.

The bacteria grew well with glycerol as only carbon source (FIG. 127A)and significant production of isoprene was demonstrated (FIG. 127B).This shows that common alcohols may also be used as carbon sources forproduction of isoprene via the mevalonate acid pathway.

Example 15 Expression of Isoprene-Synthase from Plant in Streptomycessp.

The gene for isoprene synthase Kudzu was obtained from plasmidpJ201:19813. Plasmid pJ201:19813 encodes isoprene synthase from Pueraialobata (Kudzu plant) and was codon-optimized for Pseudomonasfluorescens, Pseudomonas putida, Rhodopseudomonas palustris andCorynebacterium (FIGS. 137A-137C (SEQ ID NO:137)). Digestion of plasmidpJ201:19813 with restriction enzymes NdeI and BamHI liberated geneiso19813 that was ligated into the Streptomyces-E. coli shuttle vectorpUWL201PW (Doumith et al., Mol. Gen. Genet. 264: 477-485, 2000; FIG.129) to generate pUWL201_iso. Successful cloning was verified byrestriction analysis of pUWL201_iso. Expression of isoprene synthaseiso19813 was under control of the erm-promoter which allows forconstitutive expression in Streptomycetes species, but not forexpression in E. coli.

PUWL201PW (no insert) and pUWL201_iso were introduced in Streptomycesalbus J1074 (Sanchez et al., Chem. Biol. 9:519-531, 2002) bytransformation of protoplasts as described by Hopwood et al., The Johninnes foundation, Norwich, 1985.

A 200 μl aliquot of protoplast suspensions was transformed with 1.9 μgpUWL201PW or 2.9 μg pUWL201_iso. After incubation overnight at 28° C. onnon-selective R5-agarplates, positive transformants were selected byfurther incubation for 4 days in R3-overlay agar containing thiostrepton(250 μg/ml). Thiostrepton resistant transformants were examined forpresence of the pUWL-plasmids by plasmid preparation using Plasmid MiniKit (Qiagen). Prepared plasmid DNA was reintroduced in E. coli DH5α togenerate sufficient amounts of plasmid DNA to be analyzed by restrictionanalysis. Positive transformants were selected on ampicillin-containingL-agar plates and insert analysis was done by digestion of plasmid DNAwith NdeI and BamHI endonucleases. Isoprene synthase was identified as a1.7 kb fragment in positive pUWL201 iso clones while in the controlstrains (pUWL201PW) no such fragment was observed.

Wild type strain and transformants of S. albus containing controlplasmid pUWL201PW or isoprene synthase encoding pUWL201_iso wereanalyzed for isoprene formation. Strains were cultivated in duplicate onsolid media (tryptic soy broth agar, TSB; 2.5 ml) in presence or absenceof thiostrepton (200 μg/ml) and incubated for 4 days at 28° C. in sealedhead-space vials (total volume 20 ml). 500 μl head-space samples (endpoint measurements) were analyzed by GC-MS in SIM-mode and isoprene wasidentified according to reference retention times and molecular masses(67 m/z). Isoprene present in head-space samples was quantified bypreviously generated calibration curves. While wild-type S. albus andcontrol strains harboring pUWL201PW produced isoprene in concentrationsslightly higher than the detection limit (0.04-0.07 ppm), S. albusharboring pUWL201_iso produced isoprene in at least tenfold excesscompared to controls (0.75 ppm; FIG. 130). The results demonstratesuccessful expression of plant-derived isoprene synthase in aprokaryotic organism of the Actinomycetes group.

Example 16 Production of Isoprene or Mevalonate from Fatty Acid or PalmOil in E. coli fadR atoC LS5218 Containing the Upper or Upper and LowerMevalonic Acid Pathway Plus Kudzu Isoprene Synthase

Escherichia coli fadR atoC strain LS5218 (#6966) was obtained from theColi Genetic Stock Center. FadR encodes a transcription repressor thatnegatively regulates expression of the genes encoding fatty aciddegradation enzymes (Campbell et al., J. Bacteriol. 183: 5982-5990,2001). AtoC is a response regulator in a two-component regulatory systemwherein AtoS regulates acetolactate metabolism. The fadR atoC strainallows constitutive expression of the fatty acid degradation genes andincorporates long chain fatty acids into long-chain-lengthpolyhydroxyalkanoates. When palm oil is used as a carbon source foreither mevalonate or isoprene production, the palm oil was converted toglycerol plus fatty acid. Methods for this are well known in the art,and it can be done either enzymatically by incubation with a lipase (forexample Porcine pancreatic lipase, Candida rugosa lipase, or othersimilar lipases) or chemically by saponification with a base such assodium hydroxide.

i) E. coli fadR atoC Strain Expressing the Upper Mevalonic Acid Pathway

Strain WW4 was created by electroporating pCLPtrcUpperPathway intoLS5218 using standard methods (Sambrooke et al., Molecular Cloning: ALaboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989). Incorporationof the plasmid was demonstrated by the production of mevalonic acid(MVA) when cells were cultured in TM3 medium supplemented with eitherC12 fatty acid (FA) or palm oil as the carbon source. To demonstrateproduction of MVA by WW4 from fatty acid, cells from an overnightculture were diluted 1 to 100 into 5 mL of modified TM3 medium (TM3without yeast extract) supplemented with 0.25% C12 FA (Sigma cat#L9755). The first sign of MVA production (24 mg/L) was apparent afterovernight incubation at 30° C. of the IPTG induced culture. Productionincreased over three days with the final level of 194 mg/L of MVAproduced. To demonstrate production of MVA by WW4 from oil, cells froman overnight culture were diluted 1 to 100 into modified TM3 mediumsupplemented with 200 mg of digested palm oil per 5 mL of TM3 medium.The first sign of MVA production (50 mg/L) was apparent after overnightincubation of the IPTG induced culture at 30° C. Production increasedover three days with a final level of 500 mg/L of MVA produced.

ii) E. coli fadR atoC Strain Expressing the Upper and Lower MVA PathwayPlus Kudzu Isoprene Synthase

Escherichia coli strain WW4 (LS5218 fadR atoC pCLPtrcUpperPathway) wastransformed with pMCM118 [pTrcKKDyIkIS] to yield WW10. The incorporationof the plasmid was demonstrated by evidence of production of isoprenewhen the strain was cultured in TM3 and glucose and induced with IPTG(100, 300, or 900 μM). The strain was relatively sensitive to IPTG andshowed a significant growth defect even at 100 μM IPTG. These resultsare shown in FIG. 128A.

To test isoprene production from dodecanoic acid, WW10 was culturedovernight in L broth containing spectinomycin (50 μg/ml), and kanamycin(50 μg/ml) at 37 C with shaking at 200 rpm. The cells were washed withmodified TM3 medium by centrifugation and resuspension in their originalculture volume with this medium. The washed and resuspended cells fromthis starter culture were diluted 1 to 100 and 1 to 10 into 5 mL ofmodified TM3 medium containing 0.125% C12 Fatty Acid (Sigma cat #L9755).

To demonstrate production of mevalonate from palm oil, the oil waspredigested with lipase at 37° C. and 250 rpm for several days torelease the fatty acids (evidence of hydrolysis was judged by the foamformed when tubes were shaken).

In addition, a culture was set up by diluting the washed cells at 1 to10 into modified TM3 medium contained in test tubes with palm oil. Afurther tube was set up by the addition of 0.125% C12FA to the remainder(2.5 mL) of the washed cells without further dilution (bioconversion).After 3.75 hours of growth at 30° C. with shaking at 250 rpm all of thecultures were induced by the addition of 50 μM IPTG. Incubation wascontinued for 4 hours after which time 200 μL of each of the cultureswas assayed for isoprene accumulation with a modified head space assay(1 hour accumulation at 30° C. with shaking at 500 rpm). An additionalisoprene assay was conducted by a 12 hour incubation of the assay glassblock prior to GCMS analysis. Incubation of the induced cultures wascontinued overnight and 200 μL aliquots were again assayed for isopreneproduction (1 hour, 30 deg, 500 rpm Shel-Lab shaker) the followingmorning. Analysis of these cultures showed the production of significantlevels of isoprene. The highest levels of isoprene were observed in theculture which was seeded at 1/10 dilution from the overnight starterculture after it had been incubated and induced overnight. This resultsuggests that this culture continued to grow and increase in celldensity. These results are shown in FIG. 128B. Cell density could not bemeasured directly because the fatty acid suspension had a turbidappearance. Cell density of this culture was therefore determined byplating an aliquot of the culture and showed 8×10⁷ colony forming units.This corresponds approximately to an OD₆₀₀ of 0.1. Nevertheless, thisculture provided significant isoprene production; no isoprene isobserved for similar strains without the pathway described in thisexample.

Example 17 Improvement of Isoprene Production by Constitutive Expressionof ybhE in E. coli

This example shows production of isoprene in a strain constitutivelyexpressing ybhE (pgl) compared to a control strain with wild type ybhE.The gene ybhE (pgl) encodes a 6-phosphogluconolactonase that suppressesposttranslational gluconylation of heterologously expressed proteins andimproves product solubility and yield while also improving biomass yieldand flux through the pentose phosphate pathway (Aon et al. Applied andEnvironmental Microbiology, 74(4): 950-958, 2008).

The BL21 strain of E. coli producing isoprene (EWL256) was constructedwith constitutive expression of the ybhE gene on a replicating plasmidpBBR1MCS5 (Gentamycin) (obtained from Dr. K. Peterson, Louisiana StateUniversity).

FRT-based recombination cassettes, and plasmids for Red/ET-mediatedintegration and antibiotic marker loopout were obtained from GeneBridges GmbH (Germany). Procedures using these materials were carriedout according to Gene Bridges protocols. Primers Pgl-F and PglGI1.5-Rwere used to amplify the resistance cassette from the FRT-gb2-Cm-FRTtemplate using Stratagene Herculase II Fusion kit according to themanufacturer's protocol. The PCR reaction (50 μL final volume)contained: 5 μL buffer, 1 μL template DNA (FRT-gb2-Cm-F from GeneBridges), 10 pmols of each primer, and 1.5 μL 25 mM dNTP mix, made to 50μL with dH₂O. The reaction was cycled as follows: 1×2 minutes, 95° C.then 30 cycles of (30 seconds at 95° C.; 30 seconds at 63° C.; 3 minutesat 72° C.).

The resulting PCR product was purified using the QiaQick PCRpurification kit (Qiagen) and electroporated into electrocompetentMG1655 cells harboring the pRed-ET recombinase-containing plasmid asfollows. Cells were prepared by growing in 5 mLs of L broth to andOD600˜0.6 at 30° C. The cells were induced for recombinase expression bythe addition of 4% arabinose and allowed to grow for 30 minutes at 30°C. followed by 30 minutes of growth at 37° C. An aliquot of 1.5 mLs ofthe cells was washed 3-4 times in ice cold dH₂O. The final cell pelletwas resuspended in 40 μL of ice cold dH₂O and 2-5 μL of the PCR productwas added. The electroporation was carried out in 1-mm gap cuvettes, at1.3 kV in a Gene Pulser Electroporator (Bio-Rad Inc.). Cells wererecovered for 1-2 hours at 30° C. and plated on L agar containingchloramphenicol (5 μg/mL). Five transformants were analyzed by PCR andsequencing using primers flanking the integration site (2 primer sets:pgl and 49 rev and 3′ EcoRV-pglstop; Bottom Pgb2 and Top GB's CMP(946)). A correct transformant was selected and this strain wasdesignated MG1655 GI1.5-pgl::CMP.

The chromosomal DNA of MG1655 GI1.5-pgl::CMP was used as template togenerate a PCR fragment containing the FRT-CMP-FRT-GI1.5-ybhE construct.This construct was cloned into pBBR1MCS5 (Gentamycin) as follows. Thefragment, here on referred to as CMP-GI1.5-pgl, was amplified using the5′ primer Pglconfirm-F and 3′ primer 3′ EcoRV-pglstop. The resultingfragment was cloned using the Invitrogen TOPO-Blunt cloning kit into theplasmid vector pCR-Blunt II-TOPO as suggested from the manufacturer. TheNsiI fragment harboring the CMP-GI1.5-pgl fragment was cloned into thePstI site of pBBR1MCS5 (Gentamycin). A 20 μl ligation reaction wasprepared containing 5 μl CMP-GI1.5-pgl insert, 2 μl pBBR1MCS5(Gentamycin) vector, 1 μl T4 DNA ligase (New England Biolabs), 2 μl 10×ligase buffer, and 10 μl ddH₂O. The ligation mixture was incubated atroom temperature for 40 minutes then 2-4 μL were electroporated intoelectrocompetent Top10 cells (Invitrogen) using the parameters disclosedabove. Transformants were selected on L agar containing 10 μg/mlchloramphenicol and 5 μg/ml Gentamycin. The sequence of the selectedclone was determined using a number of the primers described above aswell as with the in-house T3 and Reverse primers provided by Sequetech,Calif. This plasmid was designated pBBRCMPGI1.5-pgl (FIGS. 135A-B andSEQ ID NO:136).

Plasmid pBBRCMPGI1.5-pgl was electroporated into EWL256, as describedabove in Example 10 and transformants were plated on L agar containingChloramphenicol (10 μg/mL), Gentamycin (5 μg/mL), spectinomycin (50μg/mL), and carbenicillin (50 μg/mL). One transformant was selected anddesignated RM11608-2.

Primers: Pgl-F (SEQ ID NO: 129)5′-ACCGCCAAAAGCGACTAATTTTAGCTGTTACAGTCAGTTGAATTAACCCTCACTAAAGGGCGGCCGC-3′ PglGI1.5-R (SEQ ID NO: 130)5′-GCTGGCGATATAAACTGTTTGCTTCATGAATGCTCCTTTGGGTTACCTCCGGGAAACGCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGGATGTGGCATAGTCAAGGGCGTGACGGCTCGCTAATACGACTCACTATAGGGC TCGAG-3′ 3′EcoRV-pglstop: (SEQ ID NO: 131)5′-CTT GAT ATC TTA GTG TGC GTT AAC CAC CAC pgl +49 rev: (SEQ ID NO: 132)CGTGAATTTGCTGGCTCTCAG Bottom Pgb2: (SEQ ID NO: 133)GGTTTAGTTCCTCACCTTGTC Top GB's CMP (946): (SEQ ID NO: 134)ACTGAAACGTTTTCATCGCTC Pglconfirm-F (SEQ ID NO: 135)5′-ACCGCCAAAAGCGACTAATTTTAGCT-3′i) Small Scale AnalysisMedia Recipe (Per Liter Fermentation Media):

K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g,1000× Trace Metals Solution 1 ml. All of the components were addedtogether and dissolved in diH₂O. The pH was adjusted to 6.8 withammonium hydroxide (30%) and brought to volume. Media wasfilter-sterilized with a 0.22 micron filter. Glucose 5.0 g andantibiotics were added after sterilization and pH adjustment.

1000× Trace Metal Solution (Per Liter Fermentation Media):

Citric Acid*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄.7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in diH₂O.The pH is adjusted to 3.0 with HCl/NaOH, and then the solution isbrought to volume and filter-sterilized with a 0.22 micron filter.

a) Experimental Procedure

Isoprene production was analyzed by growing the strains in a Cellerator™from MicroReactor Technologies, Inc. The working volume in each of the24 wells was 4.5 mL. The temperature was maintained at 30° C., the pHsetpoint was 7.0, the oxygen flow setpoint was 20 sccm and the agitationrate was 800 rpm. An inoculum of E. coli strain taken from a frozen vialwas streaked onto an LB broth agar plate (with antibiotics) andincubated at 30° C. A single colony was inoculated into media withantibiotics and grown overnight. The bacteria were diluted into 4.5 mLof media with antibiotics to reach an optical density of 0.05 measuredat 550 nm.

Off-gas analysis of isoprene was performed using a gaschromatograph-mass spectrometer (GC-MS) (Agilent) headspace assay.Sample preparation was as follows: 100 μL of whole broth was placed in asealed GC vial and incubated at 30° C. for a fixed time of 30 minutes.Following a heat kill step, consisting of incubation at 70° C. for 5minutes, the sample was loaded on the GC.

Optical density (OD) at a wavelength of 550 nm was obtained using amicroplate reader (Spectramax) during the course of the run. Specificproductivity was obtained by dividing the isoprene concentration (μg/L)by the OD reading and the time (hour).

The two strains EWL256 and RM11608-2 were assessed at 200 and 400 μMIPTG induction levels. Samples were analyzed for isoprene production andcell growth (OD₅₅₀) at 1, 2.5, 4.75, and 8 hours post-induction. Sampleswere done in duplicate.

b) Results

The experiment demonstrated that at 2 different concentrations of IPTGthe strain expressing the ybhE (pgl) had a dramatic 2-3 fold increase inspecific productivity of isoprene compared to the control strain.

ii) Isoprene fermentation from E. coli expressing M. mazei mevalonatekinase, P. alba isoprene synthase, and pgl over-expression (RHM111608-2)and grown in fed-batch culture at the 15-L scale

Medium Recipe (Per Liter Fermentation Medium)

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized witha 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCL Upper), theintegrated lower MVA pathway (gi1.2KKDyI), high expression of mevalonatekinase from M. mazei and isoprene synthase from P. alba (pTrcAlba-mMVK),and high expression of pgl (pBBR-pgl). This experiment was carried outto monitor isoprene formation from glucose at the desired fermentationpH 7.0 and temperature 34° C. A frozen vial of the E. coli strain wasthawed and inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0, measured at 550 nm, 500 mL was used toinoculate a 15-L bioreactor bringing the initial volume to 5-L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 40 hour (59 hour) fermentation was 3.1 kg (4.2 kgat 59 hour). Induction was achieved by adding IPTG. The IPTGconcentration was brought to 110 μM when the optical density at 550 nm(OD₅₅₀) reached a value of 4. The IPTG concentration was raised to 192μM when OD₅₅₀ reached 150. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 136A. The isoprene level in the off gas from thebioreactor was determined using a Hiden mass spectrometer. The isoprenetiter increased over the course of the fermentation to a maximum valueof 33.2 g/L at 40 hours (48.6 g/L at 59 hours) (FIG. 136B). The isoprenetiter increased over the course of the fermentation to a maximum valueof 40.0 g/L at 40 hours (60.5 g/L at 59 hours) (FIG. 136C). The totalamount of isoprene produced during the 40-hour (59-hour) fermentationwas 281.3 g (451.0 g at 59 hours) and the time course of production isshown in FIG. 136D. The time course of volumetric productivity is shownin FIG. 136E and shows that an average rate of 1.0 g/L/hr was maintainedbetween 0 and 40 hours (1.4 g/L/hour between 19 and 59 hour). Themetabolic activity profile, as measured by CER, is shown in FIG. 136F.The molar yield of utilized carbon that went into producing isopreneduring fermentation was 19.6% at 40 hours (23.6% at 59 hours). Theweight percent yield of isoprene from glucose was 8.9% at 40 hours(10.7% at 59 hours)

Example 18 Isoprene Polymerization

Preparation of Isoprene Samples for Polymerization

(a) Preparation of 1000× Modified Trace Metal Solution:

Each of the following components is dissolved one at a time in Di H₂O:Citric Acid*H₂O (40 g), MnSO₄*H₂O (30 g), NaCl (10 g), FeSO₄*7H₂O (1 g),CoCl₂*6H₂O (1 g), ZnSO*7H₂O (1 g), CuSO₄*5H₂O (100 mg), H₃BO₃ (100 mg),NaMoO₄*2H₂O (100 mg). The pH was adjusted to 3.0 with HCl/NaOH, thenq.s. to volume and filter sterilized with a 0.22 micron filter.

(b) Preparation of Fermentation Medium:

Each liter of fermentation medium contained K₂HPO₄ (7.5 g), MgSO₄*7H₂O(2 g), citric acid monohydrate (2 g), ferric ammonium citrate (0.3 g),yeast extract (0.5 g), 1000× Modified Trace Metal Solution (1 ml). Allof the components were added together and dissolved in diH2O. Thissolution was autoclaved. The pH was adjusted to 7.0 with ammonium gas(NH₃) and q.s. to volume. Glucose (10 g), thiamine*HCl (0.1 g), andantibiotic were added after sterilization and pH adjustment.

(c) Collection of Isoprene Samples for Purification and Polymerization:

Isoprene was collected by adsorption on activated charcoal by passingthe fermentation exhaust across canisters of activated charcoal arrangedin parallel on an exhaust manifold.

(d) Preparation of Isoprene Polymerization Sample a from Glucose UsingE. coli

Fermentation was performed at pH 7.0 and 30° C. in a 15-L bioreactorwith BL21 (DE3) E. coli cells containing the pCL PtrcUpperMVA and pTrcKKDyIkIS plasmids. An inoculum of E. coli strain taken from a frozenvial was streaked onto an LB broth agar plate (with antibiotics) andincubated at 37° C. A single colony was inoculated into tryptone-yeastextract medium. After the inoculum grew to OD 1.0, measured at 550 nm,500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 54 hour fermentation was 3.7 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 25 μM when the optical density at 550nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to50 μM when OD550 reached 190. IPTG concentration was raised to 100 μM at38 hours of fermentation. The OD550 profile within the bioreactor overtime is shown in FIG. 138. The isoprene titer increased over the courseof the fermentation to a final value of 2.2 g/L (FIG. 139). The totalamount of isoprene produced during the 54 hour fermentation was 15.9 gand the time course of production is shown in FIG. 140. The molar yieldof utilized carbon that went into producing isoprene during fermentationwas 1.53%. (See FIGS. 138-140).

(e) Preparation of Isoprene Polymerization Sample B from Glucose andYeast Extract Using E. coli

Isoprene formation from glucose and yeast extract was performed at pH7.0 and 30° C. in a 500-L bioreactor with E. coli cells containing thepTrcKudzu+yIDI+DXS plasmid. An inoculum of E. coli strain taken from afrozen vial was prepared in soytone-yeast extract-glucose medium. Afterthe inoculum grew to OD 0.15, measured at 550 nm, 20 mL was used toinoculate a bioreactor containing 2.5-L tryptone-yeast extract medium.The 2.5-L bioreactor was grown at 30° C. to OD 1.0 and 2.0-L wastransferred to the 500-L bioreactor. Yeast extract (Bio Springer,Montreal, Quebec, Canada) and glucose were fed at exponential rates. Thetotal amount of glucose and yeast extract delivered to the bioreactorduring the 50 hour fermentation was 181.2 kg and 17.6 kg, respectively.The optical density within the bioreactor over time is shown in FIG.141. The isoprene titer increased over the course of the fermentation(FIG. 142). The total amount of isoprene produced during the 50 hourfermentation was 55.1 g and the time course of production is shown inFIG. 143.

Isoprene Desorption from Activated Charcoal (Method A):

Activated charcoal (130 g), which had been exposed to a stream offermentor off-gas, was placed into a 1000 mL flask along with a stirbar. Cyclohexane (563 mL) was added to the flask and the slurry wasagitated for 2 hours. Vacuum was applied (100 mbar) via an in-linecryogenic trap (30 mL capacity, immersed in liq. nitrogen). Fourfractions were collected and combined to yield an isoprene/cyclohexanesolution (2.1 wt % isoprene, total volume=53.1 g). This solution wasvacuum distilled at 100 mbar and a new isoprene/cyclohexane solution wascollected (yield=10.1 g), which was dried over 3 A molecular sieves. GCanalysis of this solution indicated an isoprene content of 7.7 wt. %.

Isoprene Desorption from Activated Charcoal (Method B):

Activated charcoal (65 g), which had been exposed to a stream offermentor off-gas, was placed into a 500 mL flask along with a stir bar.Jarytherm DBT (250 g) was added to the charcoal and the slurry wasagitated for 2 hours. Vacuum was applied (5 mbar) via an in-linecryogenic trap (30 mL capacity, immersed in liq. nitrogen). After 1 hourthe trap was warmed to ambient temperature. Two liquid phases werepresent in the trap (total weight 1.82 g). The organic phase was dilutedwith cyclohexane (3.26 g), decanted, and dried over 3 A molecularsieves. GC analysis of this solution indicated an isoprene content of27.3 wt. %, or 1.22 g).

Preparation of Neodymium Catalyst:

Neodymium versatate (2.68 mL, 0.51 M in hexane,), triisobutylaluminum(54 mL, 1.0 M in hexane), and diethylaluminum chloride (3.40 mL, 1.0 Min hexane) were drawn up into plastic syringes fitted with steelcannula. The first two components were added to a solution of1,3-butadiene in hexane (22.4 mL, 15 wt. % 1,3-butadiene, placed into a100 mL glass vessel with septum top, and agitated for 0.5 h at ambienttemperature. The last component was added to the solution after which itwas heat-aged for 0.5 h at 65° C. The final solution was clear andyellow. The concentration of the solution based on neodymium was 0.0164M.

Preparation of Titanium Catalyst:

A 100 mL glass reaction vessel with septum inlet and containing amagnetic stirbar was placed in an ice bath at 0° C., charged withn-hexane (5.07 mL, anhydrous), and with neat TiCl₄ (1.5 mL, 13.7 mmol)under vigorous agitation. Separately, a solution was generatedconsisting of diphenyl ether (1.2 mL, 7.6 mmol) and triisobutylaluminum(14.6 mL, 12.6 mmol, 25 wt. % solution in hexane). The solution wasadded to the reaction vessel over the course of 5 minutes. A brownprecipitate formed during the addition. The suspension was stirred for10 minutes and was then stored at −40° C. for future use.

Polymerization:

Samples of polyisoprene derived primarily from glucose were produced bypolymerizing Isoprene Polymerization Sample A with Neodymium catalystand n-BuLi. Samples of polyisoprene derived from cofermentation ofglucose and yeast extract were produced by polymerizing IsoprenePolymerization Sample B with Neodymium catalyst, titatium catalyst,n-BuLi catalyst, and emulsion free radical polymerization.Representative polymerization conditions are described below.

Solution Polymerization of Isoprene with Neodymium Catalyst:

A 4 mL screw top glass vial with Teflon coated stir bar was annealed inan oven for 3 h at 150° C. The vial was fitted with a Teflon facedsilicone septum and open-top cap. Using a syringe, it was then chargedwith an isoprene solution (1.5 g, 7.7 wt. % in cyclohexane, anhydrous).Neodymium catalyst solution (60 μL) was injected into the vial with amicro-syringe. The vial was placed onto a stirrer/hotplate regulated to65° C., with the stir bar spinning at 500 rpm. After 15 minutes thesolution became noticeably more viscose. After a reaction time of 1.5 hthe reaction was quenched with a solution of isopropanol and butylatedhydroxytoluene, (BHT), (30 μL, 10 wt. % BHT). A 100 mg sample of thecement was removed for GPC analysis. The remaining polymer cement wasdried under ambient conditions. The isolated polymer weighed 110 mg, wasdetermined to have a weight average molecular weight of 935,000 (by GPC)and a cis-mirrostructure content of greater than 90% (by ¹³C-NMR).

Solution Polymerization of Isoprene with Ti Catalyst:

A 4 mL screw top glass vial and Teflon coated stir bar was annealed inan oven for 3 h at 150° C. The vial was fitted with a pre-scored Teflonfaced silicone septum and open-top cap. Using a syringe, it was thencharged with an isoprene solution (1.5 g, 7.7 wt. % in cyclohexane,anhydrous). The titanium catalyst suspension was magnetically stirredand a sample was removed (70 μL) with a disposable tip pipette, whichwas then added to the reaction vial through the pre-scored septum. Thereaction vial septum was replaced with a solid cap, and the vial wasplaced onto a stirrer/hotplate regulated to 65° C., with the stir barspinning at 500 rpm. After 5 minutes the solution became noticeably moreviscose. After a reaction time of 1.5 h the reaction was quenched with asolution of isopropanol and butylated hydroxytoluene, (BHT), (30 μL. 10wt. % BHT). A 100 mg sample of the cement was removed for GPC analysis.The remaining polymer cement was dried under ambient conditions. Theisolated polymer weighed 108 mg, had a weight average molecular weightof 221,000 (by GPC), and had a cis-mirocstructure content of greaterthan 94% (by ¹³C-NMR).

Emulsion Polymerization of Isoprene:

A 20 mL vial was used as a polymerization vessel. The metal cap waspierced twice with an awl and cardboard linear was replaced with arubber gasket and Teflon linear. The vial was rinsed with deionizedwater and dried under nitrogen.

To the vial was added 7.05 g deionized water, 1.14 g of 10% soap(potassium salt of mixed fatty acids), 174 mg of 10% ammonium persulfatesolution, and 24 mg of n-dodecane thiol. The flask was purge for 30seconds with nitrogen and capped. To the vial through the rubber/Teflongasket was charged 3 mL of bio-HG (2.033 grams of isoprene). The vialwas placed in a standard bottle polymerization bath (a second blank vialallows the vial to fit in a 4 oz bottle holder). The mixture was tumbledfor 25.5 hours at 65° C. (+/−0.2° C.).

Work-Up:

The latex was transferred to 50 mL pear shaped flask and diluted with 10mL of water. Un-reacted volatile organic was removed by evaporating 2 mLof water under vacuum (54 mmHg, 40-50° C.). To the latex was added anantioxidant dispersion, 140 mg of 50% active polyphenolic AO (Bostex24). The latex was coagulated by adding it to a dilute acid solution (12mL of 18% sulfuric acid in 150 mL RO water). The polymer coagulated intoa single large piece which was pressed and washed with RO water. Thesample was off white soft rubbery mass. The yield was 1.24 grams of wetcrumb.

The final total solids content (TSC=100*dried weight/wet weight) was18.9 wt % or an approximate conversion of 84%.

Polymerization of Isoprene with Butyllithium:

Butyllithium (1.6 M in hexane) was diluted with n-hexane (anhydrous) ina ratio of 1:10. The solution was titrated against a standardN-pivalolyl-o-benzylaniline in THF. A solution of isoprene incyclohexane (4 mL) was dried by passing it through a small columncontaining heat treated silica gel.

A 4 mL glass vial (oven dried at 150° C.) was charged with a smallTeflon coated magnetic stir bar and a solution of isoprene incyclohexane (1.35 g, 21.5 wt %). Butyllithium (0.14 M, hexane) was addedvia syringe and the vial was heated to 65° C. on a stirrer/hot plate for3 h. The polymer reaction was quenched with a BHT/iso-propanol solution(10 wt % BHT in iso-propanol). All volatiles were removed under vacuum.This procedure yielded 290 mg of polymer which represents a theoreticalyield of about 100%. This polymer was determined by GPC analysis to havea weight average molecular weight (M_(w)) of 17,880 and was determinedby ¹³C NMR to have a cis-microstructure content of 67%; atrans-microstructure content of 25%, and a 3,4-microstructure content of8%.

GPC Analysis of Polymers:

Size Exclusion Chromatography (SEC) is a well established technique tomeasure polymer molecular weight and polydispersity (Mw/Mn). Two PolymerLaboratories C microgel columns in series were utilized withtetrahydrofuran as the carrier solvent at a flow rate of 0.7 ml/min anda column temperature of 40° C. SEC was performed using a WyattTechnologies miniDawn light scattering detector coupled with a HewlettPackard 1047A refractive index detector. Polystyrene standards were usedto calibrate the instrument.

NMR Analysis of Polymers:

Polymer microstructures were determined by ¹³C-NMR analysis on a VarianUnity-Plus 400 MHz spectrometer in chloroform-d solvent.

TABLE 14 Data from ¹³C/¹²C Isotope Analyses Entry Sample (note: PI =polyisoprene) δ¹³C 1 PI from sugar beet invert sugar −34.98 2 CommercialPI from isobutylene −34.43 3 Commercial PI from isobutylene −34.42 4Guayule rubber −31.10 5 Palm oil −30.03 6 Palm oil −30.00 7 Naturalrubber (Neco) −28.11 8 Natural rubber (Pumpic) −27.92 9 Natural rubber(Negato) −27.86 10 Natural rubber (Nivco) −27.79 11 Natural rubber(Naplo) −27.74 12 Natural rubber (Krado 1) −27.68 13 Natural rubber(Krado 1) −27.55 14 Natural rubber (Krado 2) −27.54 15 Natural rubber(Krado 2) −27.52 16 Natural rubber (Krado 2) −27.49 17 Natural rubber(Nolo) −27.38 18 Yeast extract −25.70 19 Yeast extract −25.68 20Commercial PI from extractive distillation (Sample 2) −23.83 21Commercial PI from extractive distillation (Sample 2) −23.83 22 Sugarfrom softwood pulp (Sample 2) −23.25 23 Sugar from softwood pulp(Sample 1) −23.00 24 Sugar from softwood pulp (Sample 1) −22.96 25Commercial PI from extractive distillation (Sample 3) −22.95 26Commercial PI from extractive distillation (Sample 3) −22.95 27Commercial PI from extractive distillation (Sample 3) −22.94 28Commercial PI from extractive distillation (Sample 3) −22.92 29Commercial PI from extractive distillation (Sample 3) −22.90 30Commercial PI from extractive distillation (Sample 3) −22.89 31Commercial PI from extractive distillation (Sample 3) −22.89 32Commercial PI from extractive distillation (Sample 3) −22.89 33Commercial PI from extractive distillation (Sample 3) −22.87 34Commercial PI from extractive distillation (Sample 3) −22.84 35Commercial PI from extractive distillation (Sample 1) −22.63 36Commercial PI from extractive distillation (Sample 1) −22.62 37Commercial PI from extractive distillation (Sample 1) −22.54 38 PI fromIsoprene Sample B (emulsion polymerization) −19.67 39 PI from IsopreneSample B (Neodymium catalyst) −19.14 40 PI from Isoprene Sample B(Neodymium catalyst) −18.80 41 PI from Isoprene Sample B (Neodymiumcatalyst) −18.37 42 PI from Isoprene Sample B (n-BuLi catalyst) −18.1243 PI from Isoprene Sample B (n-BuLi catalyst) −18.12 44 Invert Sugar(Sample 1) −15.37 45 Invert Sugar (Sample 2) −15.36 46 Invert Sugar(Sample 1) −15.34 47 Invert Sugar (Sample 1) −15.31 48 Invert Sugar(Sample 1) −15.25 49 PI from Isoprene Sample A (Neodymium catalyst)−14.85 50 PI from Isoprene Sample A (n-BuLi catalyst) −14.69 51 PI fromIsoprene Sample A (n-BuLi catalyst) −14.69 52 PI from Isoprene Sample A(n-BuLi catalyst) −14.66 53 Glucose from bagasse (sample 2) −13.19 54Glucose from bagasse (sample 1) −13.00 55 Glucose from bagasse(sample 1) −12.93 56 Glucose from corn stover (sample 2) −11.42 57Glucose from corn stover (sample 1) −11.23 58 Glucose from corn stover(sample 1) −11.20 59 Cornstarch −11.12 60 Cornstarch −11.11 61Cornstarch −11.10 62 Cornstarch −11.07 63 Glucose −10.73

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. Singleton, et al., Dictionary ofMicrobiology and Molecular Biology, 2nd ed., John Wiley and Sons, NewYork (1994), and Hale & Marham, The Harper Collins Dictionary ofBiology, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. It is tobe understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary. Oneof skill in the art will also appreciate that any methods and materialssimilar or equivalent to those described herein can also be used topractice or test the invention.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole.

For use herein, unless clearly indicated otherwise, use of the terms“a”, “an,” and the like refers to one or more. The singular terms “a,”“an,” and “the” include the plural reference unless the context clearlyindicates otherwise

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

APPENDIX 1 Exemplary 1-deoxy-D-xylulose-5-phosphate synthase nucleicacids and polypeptides ATH: AT3G21500(DXPS1) AT4G15560(CLA1) HIN:HI1439(dxs) AT5G11380(DXPS3) HIT: NTHI1691(dxs) OSA: 4338768 43400904342614 HIP: CGSHiEE_04795 CME: CMF089C HIQ: CGSHiGG_01080 PFA:MAL13P1.186 HDU: HD0441(dxs) TAN: TA20470 HSO: HS_0905(dxs) TPV:TP01_0516 PMU: PM0532(dxs) ECO: b0420(dxs) MSU: MS1059(dxs) ECJ:JW0410(dxs) APL: APL_0207(dxs) ECE: Z0523(dxs) XFA: XF2249 ECS: ECs0474XFT: PD1293(dxs) ECC: c0531(dxs) XCC: XCC2434(dxs) ECI: UTI89_C0443(dxs)XCB: XC_1678 ECP: ECP_0479 XCV: XCV2764(dxs) ECV: APECO1_1590(dxs) XAC:XAC2565(dxs) ECW: EcE24377A_0451(dxs) XOO: XOO2017(dxs) ECX: EcHS_A0491XOM: XOO_1900(XOO1900) STY: STY0461(dxs) VCH: VC0889 STT: t2441(dxs)VVU: VV1_0315 SPT: SPA2301(dxs) VVY: VV0868 SEC: SC0463(dxs) VPA: VP0686STM: STM0422(dxs) VFI: VF0711 YPE: YPO3177(dxs) PPR: PBPRA0805 YPK:y1008(dxs) PAE: PA4044(dxs) YPM: YP_0754(dxs) PAU: PA14_11550(dxs) YPA:YPA_2671 PAP: PSPA7_1057(dxs) YPN: YPN_0911 PPU: PP_0527(dxs) YPP:YPDSF_2812 PST: PSPTO_0698(dxs) YPS: YPTB0939(dxs) PSB: Psyr_0604 YPI:YpsIP31758_3112(dxs) PSP: PSPPH_0599(dxs) SFL: SF0357(dxs) PFL:PFL_5510(dxs) SFX: S0365(dxs) PFO: Pfl_5007 SFV: SFV_0385(dxs) PEN:PSEEN0600(dxs) SSN: SSON_0397(dxs) PMY: Pmen_3844 SBO: SBO_0314(dxs)PAR: Psyc_0221(dxs) SDY: SDY_0310(dxs) PCR: Pcryo_0245 ECA: ECA1131(dxs)ACI: ACIAD3247(dxs) PLU: plu3887(dxs) SON: SO_1525(dxs) BUC: BU464(dxs)SDN: Sden_2571 BAS: BUsg448(dxs) SFR: Sfri_2790 WBR: WGLp144(dxs) SAZ:Sama_2436 SGL: SG0656 SBL: Sbal_1357 KPN: KPN_00372(dxs) SLO: Shew_2771BFL: Bfl238(dxs) SHE: Shewmr4_2731 BPN: BPEN_244(dxs) SHM: Shewmr7_2804SHN: Shewana3_2901 BPE: BP2798(dxs) SHW: Sputw3181_2831 BPA:BPP2464(dxs) ILO: IL2138(dxs) BBR: BB1912(dxs) CPS: CPS_1088(dxs) RFR:Rfer_2875 PHA: PSHAa2366(dxs) POL: Bpro_1747 PAT: Patl_1319 PNA:Pnap_1501 SDE: Sde_3381 AJS: Ajs_1038 PIN: Ping_2240 MPT: Mpe_A2631 MAQ:Maqu_2438 HAR: HEAR0279(dxs) MCA: MCA0817(dxs) MMS: mma_0331 FTU:FTT1018c(dxs) NEU: NE1161(dxs) FTF: FTF1018c(dxs) NET: Neut_1501 FTW:FTW_0925(dxs) NMU: Nmul_A0236 FTL: FTL_1072 EBA: ebA4439(dxs) FTH:FTH_1047(dxs) AZO: azo1198(dxs) FTA: FTA_1131(dxs) DAR: Daro_3061 FTN:FTN_0896(dxs) TBD: Tbd_0879 NOC: Noc_1743 MFA: Mfla_2133 AEH: Mlg_1381HPY: HP0354(dxs) HCH: HCH_05866(dxs) HPJ: jhp0328(dxs) CSA: Csal_0099HPA: HPAG1_0349 ABO: ABO_2166(dxs) HHE: HH0608(dxs) AHA: AHA_3321(dxs)HAC: Hac_0968(dxs) BCI: BCI_0275(dxs) WSU: WS1996 RMA: Rmag_0386 TDN:Tmden_0475 VOK: COSY_0360(dxs) CJE: Cj0321(dxs) NME: NMB1867 CJR:CJE0366(dxs) NMA: NMA0589(dxs) CJJ: CJJ81176_0343(dxs) NMC: NMC0352(dxs)CJU: C8J_0298(dxs) NGO: NGO0036 CJD: JJD26997_1642(dxs) CVI:CV_2692(dxs) CFF: CFF8240_0264(dxs) RSO: RSc2221(dxs) CCV:CCV52592_1671(dxs) CCV52592_1722 REU: Reut_A0882 CHA: CHAB381_1297(dxs)REH: H16_A2732(dxs) CCO: CCC13826_1594(dxs) RME: Rmet_2615 ABU:Abu_2139(dxs) BMA: BMAA0330(dxs) NIS: NIS_0391(dxs) BMV:BMASAVP1_1512(dxs) SUN: SUN_2055(dxs) BML: BMA10299_1706(dxs) GSU:GSU0686(dxs-1) GSU1764(dxs-2) BMN: BMA10247_A0364(dxs) GME: Gmet_1934Gmet_2822 BXE: Bxe_B2827 PCA: Pcar_1667 BUR: Bcep18194_B2211 PPD:Ppro_1191 Ppro_2403 BCN: Bcen_4486 DVU: DVU1350(dxs) BCH: Bcen2424_3879DVL: Dvul_1718 BAM: Bamb_3250 DDE: Dde_2200 BPS: BPSS1762(dxs) LIP:LI0408(dsx) BPM: BURPS1710b_A0842(dxs) DPS: DP2700 BPL:BURPS1106A_A2392(dxs) ADE: Adeh_1097 BPD: BURPS668_A2534(dxs) MXA:MXAN_4643(dxs) BTE: BTH_II0614(dxs) SAT: SYN_02456 SFU: Sfum_1418 BAA:BA_4853 PUB: SAR11_0611(dxs) BAT: BAS4081 MLO: mlr7474 BCE: BC4176(dxs)MES: Meso_0735 BCA: BCE_4249(dxs) SME: SMc00972(dxs) BCZ: BCZK3930(dxs)ATU: Atu0745(dxs) BTK: BT9727_3919(dxs) ATC: AGR_C_1351 BTL:BALH_3785(dxs) RET: RHE_CH00913(dxs) BLI: BL01523(dxs) RLE: RL0973(dxs)BLD: BLi02598(dxs) BME: BMEI1498 BCL: ABC2462(dxs) BMF: BAB1_0462(dxs)BAY: RBAM_022600 BMS: BR0436(dxs) BPU: BPUM_2159 BMB: BruAb1_0458(dxs)GKA: GK2392 BOV: BOV_0443(dxs) GTN: GTNG_2322 BJA: bll2651(dxs) LMO:lmo1365(tktB) BRA: BRADO2161(dxs) LMF: LMOf2365_1382(dxs) BBT:BBta_2479(dxs) LIN: lin1402(tktB) RPA: RPA0952(dxs) LWE: lwe1380(tktB)RPB: RPB_4460 LLA: L108911(dxsA) L123365(dxsB) RPC: RPC_1149 LLC:LACR_1572 LACR_1843 RPD: RPD_4305 LLM: llmg_0749(dxsB) RPE: RPE_1067SAK: SAK_0263 NWI: Nwi_0633 LPL: lp_2610(dxs) NHA: Nham_0778 LJO: LJ0406BHE: BH04350(dxs) LAC: LBA0356 BQU: BQ03540(dxs) LSL: LSL_0209(dxs) BBK:BARBAKC583_0400(dxs) LGA: LGAS_0350 CCR: CC_2068 STH: STH1842 SIL:SPO0247(dxs) CAC: CAC2077 CA_P0106(dxs) SIT: TM1040_2920 CPE: CPE1819RSP: RSP_0254(dxsA) RSP_1134(dxs) CPF: CPF_2073(dxs) JAN: Jann_0088Jann_0170 CPR: CPR_1787(dxs) RDE: RD1_0101(dxs) RD1_0548(dxs) CTC:CTC01575 MMR: Mmar10_0849 CNO: NT01CX_1983 HNE: HNE_1838(dxs) CTH:Cthe_0828 ZMO: ZMO1234(dxs) ZMO1598(dxs) CDF: CD1207(dxs) NAR: Saro_0161CBO: CBO1881(dxs) SAL: Sala_2354 CBA: CLB_1818(dxs) ELI: ELI_12520 CBH:CLC_1825(dxs) GOX: GOX0252 CBF: CLI_1945(dxs) GBE: GbCGDNIH1_0221GbCGDNIH1_2404 CKL: CKL_1231(dxs) RRU: Rru_A0054 Rru_A2619 CHY:CHY_1985(dxs) MAG: amb2904 DSY: DSY2348 MGM: Mmc1_1048 DRM: Dred_1078SUS: Acid_1783 PTH: PTH_1196(dxs) BSU: BG11715(dxs) SWO: Swol_0582 BHA:BH2779 CSC: Csac_1853 BAN: BA4400(dxs) TTE: TTE1298(dxs) BAR:GBAA4400(dxs) MTA: Moth_1511 MPE: MYPE730 LBJ: LBJ_0917(dxs) MGA:MGA_1268(dxs) LBL: LBL_0932(dxs) MTU: Rv2682c(dxs1) Rv3379c(dxs2) SYN:sll1945(dxs) MTC: MT2756(dxs) SYW: SYNW1292(Dxs) MBO: Mb2701c(dxs1)Mb3413c(dxs2) SYC: syc1087_c(dxs) MLE: ML1038(dxs) SYF: Synpcc7942_0430MPA: MAP2803c(dxs) SYD: Syncc9605_1430 MAV: MAV_3577(dxs) SYE:Syncc9902_1069 MSM: MSMEG_2776(dxs) SYG: sync_1410(dxs) MMC: Mmcs_2208SYR: SynRCC307_1390(dxs) CGL: NCg11827(cg11902) SYX: SynWH7803_1223(dxs)CGB: cg2083(dxs) CYA: CYA_1701(dxs) CEF: CE1796 CYB: CYB_1983(dxs) CDI:DIP1397(dxs) TEL: tll0623 CJK: jk1078(dxs) GVI: gll0194 NFA:nfa37410(dxs) ANA: alr0599 RHA: RHA1_ro06843 AVA: Ava_4532 SCO:SCO6013(SC1C3.01) SCO6768(SC6A5.17) PMA: Pro0928(dxs) SMA: SAV1646(dxs1)SAV2244(dxs2) PMM: PMM0907(Dxs) TWH: TWT484 PMT: PMT0685(dxs) TWS:TW280(Dxs) PMN: PMN2A_0300 LXX: Lxx10450(dxs) PMI: PMT9312_0893 CMI:CMM_1660(dxsA) PMB: A9601_09541(dxs) AAU: AAur_1790(dxs) PMC:P9515_09901(dxs) PAC: PPA1062 PMF: P9303_15371(dxs) TFU: Tfu_1917 PMG:P9301_09521(dxs) FRA: Francci3_1326 PMH: P9215_09851 FAL: FRAAL2088(dxs)PMJ: P9211_08521 ACE: Acel_1393 PME: NATL1_09721(dxs) SEN:SACE_1815(dxs) SACE_4351 TER: Tery_3042 BLO: BL1132(dxs) BTH: BT_1403BT_4099 BAD: BAD_0513(dxs) BFR: BF0873 BF4306 FNU: FN1208 FN1464 BFS:BF0796(dxs) BF4114 RBA: RB2143(dxs) PGI: PG2217(dxs) CTR: CT331(dxs)CHU: CHU_3643(dxs) CTA: CTA_0359(dxs) GFO: GFO_3470(dxs) CMU: TC0608FPS: FP0279(dxs) CPN: CPn1060(tktB_2) CTE: CT0337(dxs) CPA: CP0790 CPH:Cpha266_0671 CPJ: CPj1060(tktB_2) PVI: Cvib_0498 CPT: CpB1102 PLT:Plut_0450 CCA: CCA00304(dxs) DET: DET0745(dxs) CAB: CAB301(dxs) DEH:cbdb_A720(dxs) CFE: CF0699(dxs) DRA: DR_1475 PCU: pc0619(dxs) DGE:Dgeo_0994 TPA: TP0824 TTH: TTC1614 TDE: TDE1910(dxs) TTJ: TTHA0006 LIL:LA3285(dxs) AAE: aq_881 LIC: LIC10863(dxs) TMA: TM1770 PMO: Pmob_1001Exemplary acetyl-CoA-acetyltransferase nucleic acids and polypeptidesHSA: 38(ACAT1) 39(ACAT2) ECX: EcHS_A2365 PTR: 451528(ACAT1) STY:STY3164(yqeF) MCC: 707653(ACAT1) 708750(ACAT2) STT: t2929(yqeF) MMU:110446(Acat1) 110460(Acat2) SPT: SPA2886(yqeF) RNO: 25014(Acat1) SEC:SC2958(yqeF) CFA: 484063(ACAT2) 489421(ACAT1) STM: STM3019(yqeF) GGA:418968(ACAT1) 421587(RCJMB04_34i5) SFL: SF2854(yqeF) XLA:379569(MGC69098) 414622(MGC81403) SFX: S3052(yqeF) 414639(MGC81256) SFV:SFV_2922(yqeF) 444457(MGC83664) SSN: SSON_2283(atoB) SSON_3004(yqeF)XTR: 394562(acat2) SBO: SBO_2736(yqeF) DRE: 30643(acat2) ECA:ECA1282(atoB) SPU: 759502(LOC759502) ENT: Ent638_3299 DME: Dmel_CG10932Dmel_CG9149 SPE: Spro_0592 CEL: T02G5.4 T02G5.7 T02G5.8(kat-1) HIT:NTHI0932(atoB) ATH: AT5G48230(ACAT2/EMB1276) XCC: XCC1297(atoB) OSA:4326136 4346520 XCB: XC_2943 CME: CMA042C CME087C XCV: XCV1401(thlA)SCE: YPL028W(ERG10) XAC: XAC1348(atoB) AGO: AGOS_ADR165C XOO:XOO1881(atoB) PIC: PICST_31707(ERG10) XOM: XOO_1778(XOO1778) CAL:CaO19.1591(erg10) VCH: VCA0690 CGR: CAGL0L12364g VCO: VC0395_0630 SPO:SPBC215.09c VVU: VV2_0494 VV2_0741 MGR: MGG_01755 MGG_13499 VVY: VVA1043VVA1210 ANI: AN1409.2 VPA: VPA0620 VPA1123 VPA1204 AFM: AFUA_6G14200AFUA_8G04000 PPR: PBPRB1112 PBPRB1840 AOR: AO090103000012 AO090103000406PAE: PA2001(atoB) PA2553 PA3454 PA3589 CNE: CNC05280 PA3925 UMA:UM03571.1 PAU: PA14_38630(atoB) DDI: DDB_0231621 PPU: PP_2051(atoB)PP_2215(fadAx) PP_3754 PFA: PF14_0484 PP_4636 TET: TTHERM_00091590TTHERM_00277470 PPF: Pput_2009 Pput_2403 Pput_3523 Pput_4498TTHERM_00926980 PST: PSPTO_0957(phbA-1) PSPTO_3164(phbA-2) TCR:511003.60 PSB: Psyr_0824 Psyr_3031 ECO: b2224(atoB) PSP:PSPPH_0850(phbA1) PSPPH_2209(phbA2) ECJ: JW2218(atoB) JW5453(yqeF) PFL:PFL_1478(atoB-2) PFL_2321 PFL_3066 ECE: Z4164(yqeF) PFL_4330(atoB-2)PFL_5283 ECS: ECs3701 PFO: Pfl_1269 Pfl_1739 Pfl_2074 Pfl_2868 ECC:c2767(atoB) c3441(yqeF) PEN: PSEEN3197 PSEEN3547(fadAx) ECI:UTI89_C2506(atoB) UTI89_C3247(yqeF) PSEEN4635(phbA) ECP: ECP_2268ECP_2857 PMY: Pmen_1138 Pmen_2036 Pmen_3597 ECV: APECO1_3662(yqeF)APECO1_4335(atoB) Pmen_3662 Pmen_3820 APECO1_43352(atoB) PAR: Psyc_0252Psyc_1169 PCR: Pcryo_0278 Pcryo_1236 Pcryo_1260 H16_A1438(phaA)H16_A1445(bktB) H16_A1528 PRW: PsycPRwf_2011 H16_A1713 H16_A1720 ACI:ACIAD0694 ACIAD1612 ACIAD2516(atoB) H16_A1887 H16_A2148 H16_B0380H16_B0381 SON: SO_1677(atoB) H16_B0406 H16_B0662 SDN: Sden_1943H16_B0668 H16_B0759 H16_B1369 H16_B1771 SFR: Sfri_1338 Sfri_2063 RME:Rmet_0106 Rmet_1357 Rmet_1362 SAZ: Sama_1375 Rmet_5156 SBL: Sbal_1495BMA: BMA1316 BMA1321(phbA) BMA1436 SBM: Shew185_1489 BMV:BMASAVP1_A1805(bktB) SBN: Sbal195_1525 BMASAVP1_A1810(phbA) SLO:Shew_1667 Shew_2858 BML: BMA10299_A0086(phbA) SPC: Sputcn32_1397BMA10299_A0091 SSE: Ssed_1473 Ssed_3533 BMN: BMA10247_1076(bktB) SPL:Spea_2783 BMA10247_1081(phbA) SHE: Shewmr4_2597 BXE: Bxe_A2273 Bxe_A2335Bxe_A2342 SHM: Shewmr7_2664 Bxe_A4255 Bxe_B0377 Bxe_B0739 SHN:Shewana3_2771 Bxe_C0332 Bxe_C0574 Bxe_C0915 SHW: Sputw3181_2704 BVI:Bcep1808_0519 Bcep1808_1717 ILO: IL0872 Bcep1808_2877 Bcep1808_3594 CPS:CPS_1605 CPS_2626 Bcep1808_4015 Bcep1808_5507 Bcep1808_5644 PHA:PSHAa0908 PSHAa1454(atoB) BUR: Bcep18194_A3629 Bcep18194_A5080PSHAa1586(atoB) Bcep18194_A5091 PAT: Patl_2923 Bcep18194_A6102Bcep18194_B0263 SDE: Sde_3149 Bcep18194_B1439 PIN: Ping_0659 Ping_2401Bcep18194_C6652 Bcep18194_C6802 MAQ: Maqu_2117 Maqu_2489 Maqu_2696Bcep18194_C6874 Maqu_3162 Bcep18194_C7118 Bcep18194_C7151 CBU: CBU_0974Bcep18194_C7332 LPN: lpg1825(atoB) BCN: Bcen_1553 Bcen_1599 Bcen_2158LPF: lpl1789 Bcen_2563 Bcen_2998 Bcen_6289 LPP: lpp1788 BCH:Bcen2424_0542 Bcen2424_1790 NOC: Noc_1891 Bcen2424_2772 Bcen2424_5368AEH: Mlg_0688 Mlg_2706 Bcen2424_6232 Bcen2424_6276 HHA: Hhal_1685 BAM:Bamb_0447 Bamb_1728 Bamb_2824 HCH: HCH_05299 Bamb_4717 Bamb_5771Bamb_5969 CSA: Csal_0301 Csal_3068 BPS: BPSL1426 BPSL1535(phbA) BPSL1540ABO: ABO_0648(fadAx) BPM: BURPS1710b_2325(bktB) MMW: Mmwyl1_0073Mmwyl1_3021 BURPS1710b_2330(phbA) Mmwyl1_3053 Mmwyl1_3097 Mmwyl1_4182BURPS1710b_2453(atoB-2) AHA: AHA_2143(atoB) BPL: BURPS1106A_2197(bktB)CVI: CV_2088(atoB) CV_2790(phaA) BURPS1106A_2202(phbA) RSO:RSc0276(atoB) RSc1632(phbA) BPD: BURPS668_2160(bktB) RSc1637(bktB)RSc1761(RS02948) BURPS668_2165(phbA) REU: Reut_A0138 Reut_A1348Reut_A1353 BTE: BTH_I2144 BTH_I2256 BTH_I2261 Reut_B4561 Reut_B4738 PNU:Pnuc_0927 Reut_B5587 Reut_C5943 Reut_C6062 BPE: BP0447 BP0668 BP2059REH: H16_A0170 H16_A0867 H16_A0868 BPA: BPP0608 BPP1744 BPP3805 BPP4216H16_A0872 H16_A1297 BPP4361 BBR: BB0614 BB3364 BB4250 BB4804 BB4947 RFR:Rfer_0272 Rfer_1000 Rfer_1871 Rfer_2273 ATU: Atu2769(atoB) Atu3475Rfer_2561 Rfer_2594 ATC: AGR_C_5022(phbA) AGR_L_2713 Rfer_3839 RET:RHE_CH04018(phbAch) POL: Bpro_1577 Bpro_2140 Bpro_3113 Bpro_4187RHE_PC00068(ypc00040) RHE_PF00014(phbAf) PNA: Pnap_0060 Pnap_0458Pnap_0867 Pnap_1159 RLE: RL4621(phaA) pRL100301 pRL120369 Pnap_2136Pnap_2804 BME: BMEI0274 BMEII0817 AAV: Aave_0031 Aave_2478 Aave_3944BMF: BAB1_1783(phbA-1) BAB2_0790(phbA-2) Aave_4368 BMS: BR1772(phbA-1)BRA0448(phbA-2) AJS: Ajs_0014 Ajs_0124 Ajs_1931 Ajs_2073 BMB:BruAb1_1756(phbA-1) BruAb2_0774(phbA- Ajs_2317 Ajs_3548 2) Ajs_3738Ajs_3776 BOV: BOV_1707(phbA-1) VEI: Veis_1331 Veis_3818 Veis_4193 OAN:Oant_1130 Oant_3107 Oant_3718 Oant_4020 DAC: Daci_0025 Daci_0192Daci_3601 Daci_5988 BJA: bll0226(atoB) bll3949 bll7400 bll7819 MPT:Mpe_A1536 Mpe_A1776 Mpe_A1869 blr3724(phbA) Mpe_A3367 BRA:BRADO0562(phbA) BRADO0983(pimB) HAR: HEAR0577(phbA) BRADO3110BRADO3134(atoB) MMS: mma_0555 BBT: BBta_3558 BBta_3575(atoB) NEU:NE2262(bktB) BBta_5147(pimB) BBta_7072(pimB) NET: Neut_0610BBta_7614(phbA) EBA: ebA5202 p2A409(tioL) RPA: RPA0513(pcaF) RPA0531RPA3715(pimB) AZO: azo0464(fadA1) azo0469(fadA2) RPB: RPB_0509 RPB_0525RPB_1748 azo2172(thlA) RPC: RPC_0504 RPC_0636 RPC_0641 RPC_0832 DAR:Daro_0098 Daro_3022 RPC_1050 RPC_2005 HPA: HPAG1_0675 RPC_2194 RPC_2228HAC: Hac_0958(atoB) RPD: RPD_0306 RPD_0320 RPD_3105 RPD_3306 GME:Gmet_1719 Gmet_2074 Gmet_2213 RPE: RPE_0168 RPE_0248 RPE_3827 Gmet_2268Gmet_3302 NWI: Nwi_3060 GUR: Gura_3043 XAU: Xaut_3108 Xaut_4665 BBA:Bd0404(atoB) Bd2095 CCR: CC_0510 CC_0894 CC_3462 DOL: Dole_0671Dole_1778 Dole_2160 Dole_2187 SIL: SPO0142(bktB) SPO0326(phbA) SPO0773ADE: Adeh_0062 Adeh_2365 SPO3408 AFW: Anae109_0064 Anae109_1504 SIT:TM1040_0067 TM1040_2790 TM1040_3026 MXA: MXAN_3791 TM1040_3735 SAT:SYN_02642 RSP: RSP_0745 RSP_1354 RSP_3184 SFU: Sfum_2280 Sfum_3582 RSH:Rsph17029_0022 Rsph17029_2401 RPR: RP737 Rsph17029_3179 Rsph17029_3921RCO: RC1134 RC1135 RSQ: Rsph17025_0012 Rsph17025_2466 RFE: RF_0163(paaJ)Rsph17025_2833 RBE: RBE_0139(paaJ) JAN: Jann_0262 Jann_0493 Jann_4050RAK: A1C_05820 RDE: RD1_0025 RD1_0201(bktB) RBO: A1I_07215RD1_3394(phbA) RCM: A1E_04760 PDE: Pden_2026 Pden_2663 Pden_2870Pden_2907 PUB: SAR11_0428(thlA) Pden_4811 Pden_5022 MLO: mlr3847 DSH:Dshi_0074 Dshi_3066 Dshi_3331 MES: Meso_3374 MMR: Mmar10_0697 PLA:Plav_1573 Plav_2783 HNE: HNE_2706 HNE_3065 HNE_3133 SME: SMa1450SMc03879(phbA) NAR: Saro_0809 Saro_1069 Saro_1222 Saro_2306 SMD:Smed_0499 Smed_3117 Smed_5094 Saro_2349 Smed_5096 SAL: Sala_0781Sala_1244 Sala_2896 Sala_3158 SWI: Swit_0632 Swit_0752 Swit_2893Swit_3602 SSP: SSP0325 SSP2145 Swit_4887 Swit_5019 LMO: lmo1414Swit_5309 LMF: LMOf2365_1433 ELI: ELI_01475 ELI_06705 ELI_12035 LIN:lin1453 GBE: GbCGDNIH1_0447 LWE: lwe1431 ACR: Acry_1847 Acry_2256 LLA:L11745(thiL) L25946(fadA) RRU: Rru_A0274 Rru_A1380 Rru_A1469 LLC:LACR_1665 LACR_1956 Rru_A1946 Rru_A3387 LLM: llmg_0930(thiL) MAG:amb0842 SPY: SPy_0140 SPy_1637(atoB) MGM: Mmc1_1165 SPZ: M5005_Spy_0119M5005_Spy_0432 ABA: Acid345_3239 M5005_Spy_1344(atoB) BSU: BG11319(mmgA)BG13063(yhfS) SPM: spyM18_0136 spyM18_1645(atoB) BHA: BH1997 BH2029BH3801(mmgA) SPG: SpyM3_0108 SpyM3_1378(atoB) BAN: BA3687 BA4240 BA5589SPS: SPs0110 SPs0484 BAR: GBAA3687 GBAA4240 GBAA5589 SPH:MGAS10270_Spy0121 BAA: BA_0445 BA_4172 BA_4700 MGAS10270_Spy0433 BAT:BAS3418 BAS3932 BAS5193 MGAS10270_Spy1461(atoB) BCE: BC3627 BC4023BC5344 SPI: MGAS10750_Spy0124 MGAS10750_Spy0452 BCA: BCE_3646 BCE_4076BCE_5475 MGAS10750_Spy1453(atoB) BCZ: BCZK3329(mmgA) BCZK3780(thl) SPJ:MGAS2096_Spy0123 MGAS2096_Spy0451 BCZK5044(atoB) MGAS2096_Spy1365(atoB)BCY: Bcer98_2722 Bcer98_3865 SPK: MGAS9429_Spy0121 MGAS9429_Spy0431 BTK:BT9727_3379(mmgA) BT9727_3765(thl) MGAS9429_Spy1339(atoB)BT9727_5028(atoB) SPF: SpyM50447(atoB2) BTL: BALH_3262(mmgA)BALH_3642(fadA) SPA: M6_Spy0166 M6_Spy0466 M6_Spy1390 BALH_4843(atoB)SPB: M28_Spy0117 M28_Spy0420 BLI: BL03925(mmgA) M28_Spy1385(atoB) BLD:BLi03968(mmgA) SAK: SAK_0568 BCL: ABC0345 ABC2989 ABC3617 LJO: LJ1609ABC3891(mmgA) LAC: LBA0626(thiL) BAY: RBAM_022450 LSA: LSA1486 BPU:BPUM_2374(yhfS) BPUM_2941 LDB: Ldb0879 BPUM_3373 LBU: LBUL_0804 OIH:OB0676 OB0689 OB2632 OB3013 LBR: LVIS_2218 GKA: GK1658 GK3397 LCA:LSEI_1787 SAU: SA0342 SA0534(vraB) LGA: LGAS_1374 SAV: SAV0354SAV0576(vraB) LRE: Lreu_0052 SAM: MW0330 MW0531(vraB) EFA: EF1364 SAR:SAR0351(thl) SAR0581 OOE: OEOE_0529 SAS: SAS0330 SAS0534 STH: STH2913STH725 STH804 SAC: SACOL0426 SACOL0622(atoB) CAC: CAC2873 CA_P0078(thiL)SAB: SAB0304(th1) SAB0526 CPE: CPE2195(atoB) SAA: SAUSA300_0355SAUSA300_0560(vraB) CPF: CPF_2460 SAO: SAOUHSC_00336 SAOUHSC_00558 CPR:CPR_2170 SAJ: SaurJH9_0402 CTC: CTC00312 SAH: SaurJH1_0412 CNO:NT01CX_0538 NT01CX_0603 SEP: SE0346 SE2384 CDF: CD1059(thlA1)CD2676(thlA2) SER: SERP0032 SERP0220 CBO: CBO3200(thl) SHA: SH0510(mvaC)SH2417 CBE: Cbei_0411 Cbei_3630 CKL: CKL_3696(thlA1) CKL_3697(thlA2)RHA1_ro03022 RHA1_ro03024 RHA1_ro03391 CKL_3698(thlA3) RHA1_ro03892 AMT:Amet_4630 RHA1_ro04599 RHA1_ro05257 RHA1_ro08871 AOE: Clos_0084Clos_0258 SCO: SCO5399(SC8F4.03) CHY: CHY_1288 CHY_1355(atoB) CHY_1604SMA: SAV1384(fadA5) SAV2856(fadA1) CHY_1738 ART: Arth_1160 Arth_2986Arth_3268 Arth_4073 DSY: DSY0632 DSY0639 DSY1567 DSY1710 NCA: Noca_1371Noca_1797 Noca_1828 DSY2402 DSY3302 Noca_2764 Noca_4142 DRM: Dred_0400Dred_1491 Dred_1784 TFU: Tfu_1520 Tfu_2394 Dred_1892 FRA: Francci3_3687SWO: Swol_0308 Swol_0675 Swol_0789 FRE: Franean1_1044 Franean1_2711Swol_1486 Swol_1934 Swol_2051 Franean1_2726 Franean1_3929 TTE:TTE0549(paaJ) Franean1_4037 Franean1_4577 MTA: Moth_1260 FAL: FRAAL2514FRAAL2618 MTU: Rv1135A Rv1323(fadA4) Rv3546(fadA5) FRAAL5910(atoB) MTC:MT1365(phbA) ACE: Acel_0626 Acel_0672 MBO: Mb1167 Mb1358(fadA4)Mb3576(fadA5) SEN: SACE_1192(mmgA) SACE_2736(fadA6) Mb3586c(fadA6)SACE_4011(catF) MBB: BCG_1197 BCG_1385(fadA4) SACE_6236(fadA4)BCG_3610(fadA5) BCG_3620c(fadA6) STP: Strop_3610 MLE: ML1158(fadA4) SAQ:Sare_1316 Sare_3991 MPA: MAP2407c(fadA3) MAP2436c(fadA4) RXY: Rxyl_1582Rxyl_1842 Rxyl_2389 Rxyl_2530 MAV: MAV_1544 MAV_1573 MAV_1863 FNU:FN0495 MAV_5081 BGA: BG0110(fadA) MSM: MSMEG_2224 MSMEG_4920 BAF:BAPKO_0110(fadA) MUL: MUL_0357 LIL: LA0457(thiL1) LA0828(thiL2)LA4139(fadA) MVA: Mvan_1976 Mvan_1988 Mvan_4305 LIC: LIC10396(phbA)Mvan_4677 Mvan_4891 LBJ: LBJ_2862(paaJ-4) MGI: Mflv_1347 Mflv_1484Mflv_2040 Mflv_2340 LBL: LBL_0209(paaJ-4) Mflv_4356 Mflv_4368 SYN:slr1993(phaA) MMC: Mmcs_1758 Mmcs_1769 Mmcs_3796 SRU: SRU_1211(atoB)SRU_1547 Mmcs_3864 CHU: CHU_1910(atoB) MKM: Mkms_0251 Mkms_1540Mkms_1805 GFO: GFO_1507(atoB) Mkms_1816 Mkms_2836 Mkms_3159 FJO:Fjoh_4612 Mkms_3286 Mkms_3869 Mkms_3938 Mkms_4227 FPS: FP0770 FP1586FP1725 Mkms_4411 Mkms_4580 RRS: RoseRS_3911 RoseRS_4348 Mkms_4724Mkms_4764 Mkms_4776 RCA: Rcas_0702 Rcas_3206 MJL: Mjls_0231 Mjls_1739Mjls_1750 Mjls_2819 HAU: Haur_0522 Mjls_3119 Mjls_3235 DRA: DR_1072DR_1428 DR_1960 DR_2480 Mjls_3800 Mjls_3850 Mjls_4110 Mjls_4383 DR_A0053Mjls_4705 Mjls_4876 DGE: Dgeo_0755 Dgeo_1305 Dgeo_1441 Mjls_5018Mjls_5063 Mjls_5075 Dgeo_1883 CGL: NCgl2309(cgl2392) TTH: TTC0191TTC0330 CGB: cg2625(pcaF) TTJ: TTHA0559 CEF: CE0731 CE2295 TME:Tmel_1134 CJK: jk1543(fadA3) FNO: Fnod_0314 NFA: nfa10750(fadA4) PMO:Pmob_0515 RHA: RHA1_ro01455 RHA1_ro01623 HMA: rrnAC0896(acaB3)rrnAC2815(aca2) RHA1_ro01876 RHA1_ro02517(catF) rrnAC3497(yqeF)rrnB0240(aca1) rrnB0242(acaB2) rrnB0309(acaB1) MSE: Msed_0656 TAC:Ta0582 PAI: PAE1220 TVO: TVN0649 PIS: Pisl_0029 Pisl_1301 PTO: PTO1505PCL: Pcal_0781 APE: APE_2108 PAS: Pars_0309 Pars_1071 SSO:SSO2377(acaB-4) CMA: Cmaq_1941 STO: ST0514 SAI: Saci_0963Saci_1361(acaB1) Exemplary HMG-CoA synthase nucleic acids andpolypeptides HSA: 3157(HMGCS1) 3158(HMGCS2) YPP: YPDSF_1517 PTR:457169(HMGCS2) 461892(HMGCS1) YPS: YPTB1475 MCC: 702553(HMGCS1)713541(HMGCS2) CBD: COXBU7E912_1931 MMU: 15360(Hmgcs2) 208715(Hmgcs1)TCX: Tcr_1719 RNO: 24450(Hmgcs2) 29637(Hmgcs1) DNO: DNO_0799 CFA:479344(HMGCS1) 607923(HMGCS2) BMA: BMAA1212 BTA: 407767(HMGCS1) BPS:BPSS1002 SSC: 397673(CH242-38B5.1) BPM: BURPS1710b_A2613 GGA:396379(HMGCS1) BPL: BURPS1106A_A1384 XLA: 380091(hmgcs1)447204(MGC80816) BPD: BURPS668_A1470 DRE: 394060(hmgcs1) BTE: BTH_II1670SPU: 578259(LOC578259) MXA: MXAN_3948(tac) MXAN_4267(mvaS) DME:Dmel_CG4311(Hmgs) BSU: BG10926(pksG) CEL: F25B4.6 OIH: OB2248 ATH:AT4G11820(BAP1) SAU: SA2334(mvaS) OSA: 4331418 4347614 SAV:SAV2546(mvaS) CME: CMM189C SAM: MW2467(mvaS) SCE: YML126C(ERG13) SAR:SAR2626(mvaS) AGO: AGOS_ADL356C SAS: SAS2432 PIC: PICST_83020 SAC:SACOL2561 CAL: CaO19_7312(CaO19.7312) SAB: SAB2420(mvaS) CGR:CAGL0H04081g SAA: SAUSA300_2484 SPO: SPAC4F8.14c(hcs) SAO: SAOUHSC_02860MGR: MGG_01026 SAJ: SaurJH9_2569 ANI: AN4923.2 SAH: SaurJH1_2622 AFM:AFUA_3G10660 AFUA_8G07210 SEP: SE2110 AOR: AO090003000611 AO090010000487SER: SERP2122 CNE: CNC05080 CNG02670 SHA: SH0508(mvaS) UMA: UM05362.1SSP: SSP0324 ECU: ECU10_0510 LMO: lmo1415 DDI: DDBDRAFT_0217522DDB_0219924(hgsA) LMF: LMOf2365_1434(mvaS) TET: TTHERM_00691190 LIN:lin1454 TBR: Tb927.8.6110 LWE: lwe1432(mvaS) YPE: YPO1457 LLA:L13187(hmcM) YPK: y2712(pksG) LLC: LACR_1666 YPM: YP_1349(pksG) LLM:llmg_0929(hmcM) YPA: YPA_0750 SPY: SPy_0881(mvaS.2) YPN: YPN_2521 SPZ:M5005_Spy_0687(mvaS.1) SPM: spyM18_0942(mvaS2) LJO: LJ1607 SPG:SpyM3_0600(mvaS.2) LAC: LBA0628(hmcS) SPS: SPs1253 LSA: LSA1484(mvaS)SPH: MGAS10270_Spy0745(mvaS1) LSL: LSL_0526 SPI:MGAS10750_Spy0779(mvaS1) LDB: Ldb0881(mvaS) SPJ: MGAS2096_Spy0759(mvaS1)LBU: LBUL_0806 SPK: MGAS9429_Spy0743(mvaS1) LBR: LVIS_1363 SPF:SpyM51121(mvaS) LCA: LSEI_1785 SPA: M6_Spy0704 LGA: LGAS_1372 SPB:M28_Spy0667(mvaS.1) LRE: Lreu_0676 SPN: SP_1727 PPE: PEPE_0868 SPR:spr1571(mvaS) EFA: EF1363 SPD: SPD_1537(mvaS) OOE: OEOE_0968 SAG:SAG1316 LME: LEUM_1184 SAN: gbs1386 NFA: nfa22120 SAK: SAK_1347 SEN:SACE_4570(pksG) SMU: SMU.943c BBU: BB0683 STC: str0577(mvaS) BGA: BG0706STL: stu0577(mvaS) BAF: BAPKO_0727 STE: STER_0621 FJO: Fjoh_0678 SSA:SSA_0338(mvaS) HAL: VNG1615G(mvaB) SSU: SSU05_1641 HMA: rrnAC1740(mvaS)SSV: SSU98_1652 HWA: HQ2868A(mvaB) SGO: SGO_0244 NPH: NP2608A(mvaB_1)NP4836A(mvaB_2) LPL: lp_2067(mvaS) Exemplary hydroxymethylglutaryl-CoAreductase nucleic acids and polypeptides HSA: 3156(HMGCR) ECU:ECU10_1720 PTR: 471516(HMGCR) DDI: DDB_0191125(hmgA) DDB_0215357(hmgB)MCC: 705479(HMGCR) TBR: Tb927.6.4540 MMU: 15357(Hmgcr) TCR: 506831.40509167.20 RNO: 25675(Hmgcr) LMA: LmjF30.3190 CFA: 479182(HMGCR) VCH:VCA0723 BTA: 407159(HMGCR) VCO: VC0395_0662 GGA: 395145(RCJMB04_14m24)VVU: VV2_0117 SPU: 373355(LOC373355) VVY: VVA0625 DME:Dmel_CG10367(Hmgcr) VPA: VPA0968 CEL: F08F8.2 VFI: VFA0841 OSA: 4347443PAT: Patl_0427 SCE: YLR450W(HMG2) YML075C(HMG1) CBU: CBU_0030 CBU_0610AGO: AGOS_AER152W CBD: COXBU7E912_0151 CGR: CAGL0L11506gCOXBU7E912_0622(hmgA) SPO: SPCC162.09c(hmg1) TCX: Tcr_1717 ANI: AN3817.2DNO: DNO_0797 AFM: AFUA_1G11230 AFUA_2G03700 CVI: CV_1806 AOR:AO090103000311 AO090120000217 SUS: Acid_5728 Acid_6132 CNE: CNF04830SAU: SA2333(mvaA) UMA: UM03014.1 SAV: SAV2545(mvaA) SAM: MW2466(mvaA)MAC: MA3073(hmgA) SAB: SAB2419c(mvaA) MBA: Mbar_A1972 SEP: SE2109 MMA:MM_0335 LWE: lwe0819(mvaA) MBU: Mbur_1098 LLA: L10433(mvaA) MHU:Mhun_3004 LLC: LACR_1664 MEM: Memar_2365 LLM: llmg_0931(mvaA) MBN:Mboo_0137 SPY: SPy_0880(mvaS.1) MTH: MTH562 SPM: spyM18_0941(mvaS1) MST:Msp_0584(hmgA) SPG: SpyM3_0599(mvaS.1) MSI: Msm_0227 SPS: SPs1254 MKA:MK0355(HMG1) SPH: MGAS10270_Spy0744 AFU: AF1736(mvaA) SPI:MGAS10750_Spy0778 HAL: VNG1875G(mvaA) SPJ: MGAS2096_Spy0758 HMA:rrnAC3412(mvaA) SPK: MGAS9429_Spy0742 HWA: HQ3215A(hmgR) SPA: M6_Spy0703NPH: NP0368A(mvaA_2) NP2422A(mvaA_1) SPN: SP_1726 TAC: Ta0406m SAG:SAG1317 TVO: TVN1168 SAN: gbs1387 PTO: PTO1143 STC: str0576(mvaA) PAB:PAB2106(mvaA) STL: stu0576(mvaA) PFU: PF1848 STE: STER_0620 TKO: TK0914SSA: SSA_0337(mvaA) RCI: RCIX1027(hmgA) RCIX376(hmgA) LPL: lp_0447(mvaA)APE: APE_1869 LJO: LJ1608 IHO: Igni_0476 LSL: LSL_0224 HBU: Hbut_1531LBR: LVIS_0450 SSO: SSO0531 LGA: LGAS_1373 STO: ST1352 EFA: EF1364 SAI:Saci_1359 NFA: nfa22110 PAI: PAE2182 BGA: BG0708(mvaA) PIS: Pisl_0814SRU: SRU_2422 PCL: Pcal_1085 FPS: FP2341 PAS: Pars_0796 MMP:MMP0087(hmgA) MMQ: MmarC5_1589 Exemplary mevalonate kinase nucleic acidsand polypeptides HSA: 4598(MVK) SCE: YMR208W(ERG12) MCC: 707645(MVK)AGO: AGOS_AER335W MMU: 17855(Mvk) PIC: PICST_40742(ERG12) RNO:81727(Mvk) CGR: CAGL0F03861g CFA: 486309(MVK) SPO: SPAC13G6.11c BTA:505792(MVK) MGR: MGG_06946 GGA: 768555(MVK) ANI: AN3869.2 DRE:492477(zgc: 103473) AFM: AFUA_4G07780 SPU: 585785(LOC585785) AOR:AO090023000793 DME: Dmel_CG33671 CNE: CNK01740 OSA: 4348331 ECU:ECU09_1780 DDI: DDBDRAFT_0168621 SAN: gbs1396 TET: TTHERM_00637680 SAK:SAK_1357(mvk) TBR: Tb927.4.4070 SMU: SMU.181 TCR: 436521.9 509237.10STC: str0559(mvaK1) LMA: LmjF31.0560 STL: stu0559(mvaK1) CBU: CBU_0608CBU_0609 STE: STER_0598 CBD: COXBU7E912_0620(mvk) SSA: SSA_0333(mvaK1)LPN: lpg2039 SSU: SSU05_0289 LPF: lpl2017 SSV: SSU98_0285 LPP: lpp2022SGO: SGO_0239(mvk) BBA: Bd1027(lmbP) Bd1630(mvk) LPL: lp_1735(mvaK1)MXA: MXAN_5019(mvk) LJO: LJ1205 OIH: OB0225 LAC: LBA1167(mvaK) SAU:SA0547(mvaK1) LSA: LSA0908(mvaK1) SAV: SAV0590(mvaK1) LSL: LSL_0685(eRG)SAM: MW0545(mvaK1) LDB: Ldb0999(mvk) SAR: SAR0596(mvaK1) LBU: LBUL_0906SAS: SAS0549 LBR: LVIS_0858 SAC: SACOL0636(mvk) LCA: LSEI_1491 SAB:SAB0540(mvaK1) LGA: LGAS_1033 SAA: SAUSA300_0572(mvk) LRE: Lreu_0915SAO: SAOUHSC_00577 PPE: PEPE_0927 SEP: SE0361 EFA: EF0904(mvk) SER:SERP0238(mvk) OOE: OEOE_1100 SHA: SH2402(mvaK1) LME: LEUM_1385 SSP:SSP2122 NFA: nfa22070 LMO: lmo0010 BGA: BG0711 LMF: LMOf2365_0011 BAF:BAPKO_0732 LIN: lin0010 FPS: FP0313 LWE: lwe0011(mvk) MMP: MMP1335 LLA:L7866(yeaG) MAE: Maeo_0775 LLC: LACR_0454 MAC: MA0602(mvk) LLM:llmg_0425(mvk) MBA: Mbar_A1421 SPY: SPy_0876(mvaK1) MMA: MM_1762 SPZ:M5005_Spy_0682(mvaK1) MBU: Mbur_2395 SPM: spyM18_0937(mvaK1) MHU:Mhun_2890 SPG: SpyM3_0595(mvaK1) MEM: Memar_1812 SPS: SPs1258 MBN:Mboo_2213 SPH: MGAS10270_Spy0740(mvaK1) MST: Msp_0858(mvk) SPI:MGAS10750_Spy0774(mvaK1) MSI: Msm_1439 SPJ: MGAS2096_Spy0753(mvaK1) MKA:MK0993(ERG12) SPK: MGAS9429_Spy0737(mvaK1) HAL: VNG1145G(mvk) SPF:SpyM51126(mvaK1) HMA: rrnAC0077(mvk) SPA: M6_Spy0699 HWA: HQ2925A(mvk)SPB: M28_Spy0662(mvaK1) NPH: NP2850A(mvk) SPN: SP_0381 PTO: PTO1352 SPR:spr0338(mvk) PHO: PH1625 SPD: SPD_0346(mvk) PAB: PAB0372(mvk) SAG:SAG1326 PFU: PF1637(mvk) TKO: TK1474 MSE: Msed_1602 RCI: LRC399(mvk)PAI: PAE3108 APE: APE_2439 PIS: Pisl_0467 HBU: Hbut_0877 PCL: Pcal_1835SSO: SSO0383 STO: ST2185 SAI: Saci_2365(mvk) Exemplary phosphomevalonatekinase nucleic acids and polypeptides HSA: 10654(PMVK) SSP: SSP2120 PTR:457350(PMVK) LMO: lmo0012 MCC: 717014(PMVK) LMF: LMOf2365_0013 MMU:68603(Pmvk) LIN: lin0012 CFA: 612251(PMVK) LWE: lwe0013 BTA:513533(PMVK) LLA: L10014(yebA) DME: Dmel_CG10268 LLC: LACR_0456 ATH:AT1G31910 LLM: llmg_0427 OSA: 4332275 SPY: SPy_0878(mvaK2) SCE:YMR220W(ERG8) SPZ: M5005_Spy_0684(mvaK2) AGO: AGOS_AER354W SPM:spyM18_0939 PIC: PICST_52257(ERG8) SPG: SpyM3_0597(mvaK2) CGR:CAGL0F03993g SPS: SPs1256 SPO: SPAC343.01c SPH: MGAS10270_Spy0742(mvaK2)MGR: MGG_05812 SPI: MGAS10750_Spy0776(mvaK2) ANI: AN2311.2 SPJ:MGAS2096_Spy0755(mvaK2) AFM: AFUA_5G10680 SPK: MGAS9429_Spy0739(mvaK2)AOR: AO090010000471 SPF: SpyM51124(mvaK2) CNE: CNM00100 SPA: M6_Spy0701UMA: UM00760.1 SPB: M28_Spy0664(mvaK2) DDI: DDBDRAFT_0184512 SPN:SP_0383 TBR: Tb09.160.3690 SPR: spr0340(mvaK2) TCR: 507913.20 508277.140SPD: SPD_0348(mvaK2) LMA: LmjF15.1460 SAG: SAG1324 MXA: MXAN_5017 SAN:gbs1394 OIH: OB0227 SAK: SAK_1355 SAU: SA0549(mvaK2) SMU: SMU.938 SAV:SAV0592(mvaK2) STC: str0561(mvaK2) SAM: MW0547(mvaK2) STL:stu0561(mvaK2) SAR: SAR0598(mvaK2) STE: STER_0600 SAS: SAS0551 SSA:SSA_0335(mvaK2) SAC: SACOL0638 SSU: SSU05_0291 SAB: SAB0542(mvaK2) SSV:SSU98_0287 SAA: SAUSA300_0574 SGO: SGO_0241 SAO: SAOUHSC_00579 LPL:lp_1733(mvaK2) SAJ: SaurJH9_0615 LJO: LJ1207 SEP: SE0363 LAC: LBA1169SER: SERP0240 LSA: LSA0906(mvaK2) SHA: SH2400(mvaK2) LSL: LSL_0683 LDB:Ldb0997(mvaK) BGA: BG0710 LBU: LBUL_0904 BAF: BAPKO_0731 LBR: LVIS_0860NPH: NP2852A LCA: LSEI_1092 SSO: SSO2988 LGA: LGAS_1035 STO: ST0978 LRE:Lreu_0913 SAI: Saci_1244 PPE: PEPE_0925 EFA: EF0902 NFA: nfa22090Exemplary diphosphomevalonate decarboxylase nucleic acids andpolypeptides HSA: 4597(MVD) SAR: SAR0597(mvaD) PTR: 468069(MVD) SAS:SAS0550 MCC: 696865(MVD) SAC: SACOL0637(mvaD) MMU: 192156(Mvd) SAB:SAB0541(mvaD) RNO: 81726(Mvd) SAA: SAUSA300_0573(mvaD) CFA: 489663(MVD)SAO: SAOUHSC_00578 GGA: 425359(MVD) SAJ: SaurJH9_0614 DME: Dmel_CG8239SAH: SaurJH1_0629 SCE: YNR043W(MVD1) SEP: SE0362 AGO: AGOS_AGL232C SER:SERP0239(mvaD) PIC: PICST_90752 SHA: SH2401(mvaD) CGR: CAGL0C03630g SSP:SSP2121 SPO: SPAC24C9.03 LMO: lmo0011 MGR: MGG_09750 LMF:LMOf2365_0012(mvaD) ANI: AN4414.2 LIN: lin0011 AFM: AFUA_4G07130 LWE:lwe0012(mvaD) AOR: AO090023000862 LLA: L9089(yeaH) CNE: CNL04950 LLC:LACR_0455 UMA: UM05179.1 LLM: llmg_0426(mvaD) DDI: DDBDRAFT_0218058 SPY:SPy_0877(mvaD) TET: TTHERM_00849200 SPZ: M5005_Spy_0683(mvaD) TBR:Tb10.05.0010 Tb10.61.2745 SPM: spyM18_0938(mvd) TCR: 507993.330511281.40 SPG: SpyM3_0596(mvaD) LMA: LmjF18.0020 SPS: SPs1257 CBU:CBU_0607(mvaD) SPH: MGAS10270_Spy0741(mvaD) CBD: COXBU7E912_0619(mvaD)SPI: MGAS10750_Spy0775(mvaD) LPN: lpg2040 SPJ: MGAS2096_Spy0754(mvaD)LPF: lpl2018 SPK: MGAS9429_Spy0738(mvaD) LPP: lpp2023 SPF:SpyM51125(mvaD) TCX: Tcr_1734 SPA: M6_Spy0700 DNO: DNO_0504(mvaD) SPB:M28_Spy0663(mvaD) BBA: Bd1629 SPN: SP_0382 MXA: MXAN_5018(mvaD) SPR:spr0339(mvd1) OIH: OB0226 SPD: SPD_0347(mvaD) SAU: SA0548(mvaD) SAG:SAG1325(mvaD) SAV: SAV0591(mvaD) SAN: gbs1395 SAM: MW0546(mvaD) SAK:SAK_1356(mvaD) SMU: SMU.937 EFA: EF0903(mvaD) STC: str0560(mvaD) LME:LEUM_1386 STL: stu0560(mvaD) NFA: nfa22080 STE: STER_0599 BBU: BB0686SSA: SSA_0334(mvaD) BGA: BG0709 SSU: SSU05_0290 BAF: BAPKO_0730 SSV:SSU98_0286 GFO: GFO_3632 SGO: SGO_0240(mvaD) FPS: FP0310(mvaD) LPL:lp_1734(mvaD) HAU: Haur_1612 LJO: LJ1206 HAL: VNG0593G(dmd) LAC:LBA1168(mvaD) HMA: rrnAC1489(dmd) LSA: LSA0907(mvaD) HWA: HQ1525A(mvaD)LSL: LSL_0684 NPH: NP1580A(mvaD) LDB: Ldb0998(mvaD) PTO: PTO0478 PTO1356LBU: LBUL_0905 SSO: SSO2989 LBR: LVIS_0859 STO: ST0977 LCA: LSEI_1492SAI: Saci_1245(mvd) LGA: LGAS_1034 MSE: Msed_1576 LRE: Lreu_0914 PPE:PEPE_0926 Exemplary isopentenyl phosphate kinases (IPK) nucleic acidsand polypeptides Methanobacterium thermoautotrophicum Picrophilustorridus DSM9790 (IG-57) gi|48477569 gi|2621082 Pyrococcus abyssigi|14520758 Methanococcus jannaschii DSM 2661 gi|1590842 Pyrococcushorikoshii OT3 gi|3258052 Methanocaldococcus jannaschii gi|1590842Archaeoglobus fulgidus DSM4304 gi|2648231 Methanothermobacterthermautotrophicus gi|2621082 Exemplary isopentenyl-diphosphateDelta-isomerase (IDI) nucleic acids and polypeptides HSA: 3422(IDI1)91734(IDI2) CGR: CAGL0J06952g PTR: 450262(IDI2) 450263(IDI1) SPO:SPBC106.15(idi1) MCC: 710052(LOC710052) 721730(LOC721730) ANI: AN0579.2MMU: 319554(Idi1) AFM: AFUA_6G11160 RNO: 89784(Idi1) AOR: AO090023000500GGA: 420459(IDI1) CNE: CNA02550 XLA: 494671(LOC494671) UMA: UM04838.1XTR: 496783(idi2) ECU: ECU02_0230 SPU: 586184(LOC586184) DDI:DDB_0191342(ipi) CEL: K06H7.9(idi-1) TET: TTHERM_00237280TTHERM_00438860 ATH: AT3G02780(IPP2) TBR: Tb09.211.0700 OSA: 43387914343523 TCR: 408799.19 510431.10 CME: CMB062C LMA: LmjF35.5330 SCE:YPL117C(IDI1) EHI: 46.t00025 AGO: AGOS_ADL268C ECO: b2889(idi) PIC:PICST_68990(IDI1) ECJ: JW2857(idi) ECE: Z4227 RCM: A1E_02555 ECS:ECs3761 RRI: A1G_04195 ECC: c3467 MLO: mlr6371 ECI: UTI89_C3274 RET:RHE_PD00245(ypd00046) ECP: ECP_2882 XAU: Xaut_4134 ECV: APECO1_3638 SIL:SPO0131 ECW: EcE24377A_3215(idi) SIT: TM1040_3442 ECX: EcHS_A3048 RSP:RSP_0276 STY: STY3195 RSH: Rsph17029_1919 STT: t2957 RSQ: Rsph17025_1019SPT: SPA2907(idi) JAN: Jann_0168 SEC: SC2979(idi) RDE: RD1_0147(idi)STM: STM3039(idi) DSH: Dshi_3527 SFL: SF2875(idi) BSU: BG11440(ypgA)SFX: S3074 BAN: BA1520 SFV: SFV_2937 BAR: GBAA1520 SSN: SSON_3042SSON_3489(yhfK) BAA: BA_2041 SBO: SBO_3103 BAT: BAS1409 SDY: SDY_3193BCE: BC1499 ECA: ECA2789 BCA: BCE_1626 PLU: plu3987 BCZ: BCZK1380(fni)ENT: Ent638_3307 BCY: Bcer98_1222 SPE: Spro_2201 BTK: BT9727_1381(fni)VPA: VPA0278 BTL: BALH_1354 VFI: VF0403 BLI: BL02217(fni) PPR:PBPRA0469(mvaD) BLD: BLi02426 PEN: PSEEN4850 BAY: RBAM_021020(fni) CBU:CBU_0607(mvaD) BPU: BPUM_2020(fni) CBD: COXBU7E912_0619(mvaD) OIH:OB0537 LPN: lpg2051 SAU: SA2136(fni) LPF: lpl2029 SAV: SAV2346(fni) LPP:lpp2034 SAM: MW2267(fni) TCX: Tcr_1718 SAR: SAR2431(fni) HHA: Hhal_1623SAS: SAS2237 DNO: DNO_0798 SAC: SACOL2341(fni) EBA: ebA5678 p2A143 SAB:SAB2225c(fni) DVU: DVU1679(idi) SAA: SAUSA300_2292(fni) DDE: Dde_1991SAO: SAOUHSC_02623 LIP: LI1134 SEP: SE1925 BBA: Bd1626 SER:SERP1937(fni-2) AFW: Anae109_4082 SHA: SH0712(fni) MXA: MXAN_5021(fni)SSP: SSP0556 RPR: RP452 LMO: lmo1383 RTY: RT0439(idi) LMF:LMOf2365_1402(fni) RCO: RC0744 LIN: lin1420 RFE: RF_0785(fni) LWE:lwe1399(fni) RBE: RBE_0731(fni) LLA: L11083(yebB) RAK: A1C_04190 LLC:LACR_0457 RBO: A1I_04755 LLM: llmg_0428(fni) SPY: SPy_0879 MUL:MUL_0380(idi2) SPZ: M5005_Spy_0685 MVA: Mvan_1582 Mvan_2176 SPM:spyM18_0940 MGI: Mflv_1842 Mflv_4187 SPG: SpyM3_0598 MMC: Mmcs_1954 SPS:SPs1255 MKM: Mkms_2000 SPH: MGAS10270_Spy0743 MJL: Mjls_1934 SPI:MGAS10750_Spy0777 CGL: NCgl2223(cgl2305) SPJ: MGAS2096_Spy0756 CGB:cg2531(idi) SPK: MGAS9429_Spy0740 CEF: CE2207 SPF: SpyM51123(fni) CDI:DIP1730(idi) SPA: M6_Spy0702 NFA: nfa19790 nfa22100 SPB: M28_Spy0665RHA: RHA1_ro00239 SPN: SP_0384 SCO: SCO6750(SC5F2A.33c) SPR:spr0341(fni) SMA: SAV1663(idi) SPD: SPD_0349(fni) LXX: Lxx23810(idi)SAG: SAG1323 CMI: CMM_2889(idiA) SAN: gbs1393 AAU: AAur_0321(idi) SAK:SAK_1354(fni) PAC: PPA2115 SMU: SMU.939 FRA: Francci3_4188 STC:str0562(idi) FRE: Franean1_5570 STL: stu0562(idi) FAL: FRAAL6504(idi)STE: STER_0601 KRA: Krad_3991 SSA: SSA_0336 SEN: SACE_2627(idiB_2)SACE_5210(idi) SGO: SGO_0242 STP: Strop_4438 LPL: lp_1732(idi1) SAQ:Sare_4564 Sare_4928 LJO: LJ1208 RXY: Rxyl_0400 LAC: LBA1171 BBU: BB0684LSA: LSA0905(idi) BGA: BG0707 LSL: LSL_0682 SYN: sll1556 LDB:Ldb0996(fni) SYC: syc2161_c LBU: LBUL_0903 SYF: Synpcc7942_1933 LBR:LVIS_0861 CYA: CYA_2395(fni) LCA: LSEI_1493 CYB: CYB_2691(fni) LGA:LGAS_1036 TEL: tll1403 LRE: Lreu_0912 ANA: all4591 EFA: EF0901 AVA:Ava_2461 Ava_B0346 OOE: OEOE_1103 TER: Tery_1589 STH: STH1674 SRU:SRU_1900(idi) CBE: Cbei_3081 CHU: CHU_0674(idi) DRM: Dred_0474 GFO:GFO_2363(idi) SWO: Swol_1341 FJO: Fjoh_0269 MTA: Moth_1328 FPS:FP1792(idi) MTU: Rv1745c(idi) CTE: CT0257 MTC: MT1787(idi) CCH: Cag_1445MBO: Mb1774c(idi) CPH: Cpha266_0385 MBB: BCG_1784c(idi) PVI: Cvib_1545MPA: MAP3079c PLT: Plut_1764 MAV: MAV_3894(fni) RRS: RoseRS_2437 MSM:MSMEG_1057(fni) MSMEG_2337(fni) RCA: Rcas_2215 HAU: Haur_4687VNG6445G(crt_2) VNG7060 VNG7149 DRA: DR_1087 HMA: rrnAC3484(idi) DGE:Dgeo_1381 HWA: HQ2772A(idiA) HQ2847A(idiB) TTH: TT_P0067 NPH:NP0360A(idiB_1) NP4826A(idiA) TTJ: TTHB110 NP5124A(idiB_2) MJA: MJ0862TAC: Ta0102 MMP: MMP0043 TVO: TVN0179 MMQ: MmarC5_1637 PTO: PTO0496 MMX:MmarC6_0906 PHO: PH1202 MMZ: MmarC7_1040 PAB: PAB1662 MAE: Maeo_1184PFU: PF0856 MVN: Mevan_1058 TKO: TK1470 MAC: MA0604(idi) RCI:LRC397(fni) MBA: Mbar_A1419 APE: APE_1765.1 MMA: MM_1764 SMR: Smar_0822MBU: Mbur_2397 IHO: Igni_0804 MTP: Mthe_0474 HBU: Hbut_0539 MHU:Mhun_2888 SSO: SSO0063 MLA: Mlab_1665 STO: ST2059 MEM: Memar_1814 SAI:Saci_0091 MBN: Mboo_2211 MSE: Msed_2136 MTH: MTH48 PAI: PAE0801 MST:Msp_0856(fni) PIS: Pisl_1093 MSI: Msm_1441 PCL: Pcal_0017 MKA:MK0776(lldD) PAS: Pars_0051 AFU: AF2287 TPE: Tpen_0272 HAL:VNG1818G(idi) VNG6081G(crt_1) Exemplary isoprene synthase nucleic acidsand polypeptides Genbank Accession Nos. AY341431 AY316691 AY279379AJ457070 AY182241

What is claimed is:
 1. A method for producing polyisoprene derived fromrenewable resources comprising: (a) initiating polymerization ofisoprene monomer in an isoprene starting composition which is derivedfrom renewable resources, wherein the isoprene starting compositionderived from renewable resources comprises greater than about 2 mg ofthe isoprene monomer and comprises one or more compounds selected fromthe group consisting of ethanol, acetone, C5 prenyl alcohols, isoprenoidcompounds with 10 or more carbon atoms, methanol, acetaldehyde,methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl,2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine; (b)allowing the polymerization of the isoprene monomer to continue toproduce the polyisoprene; (c) terminating the polymerization of theisoprene monomer, and (d) recovering the polyisoprene.
 2. The method ofclaim 1, wherein the isoprene starting composition derived fromrenewable resources comprises greater than or about 99.94% isoprene byweight compared to the total weight of all C5 hydrocarbons in thecomposition.
 3. The method of claim 1, wherein the isoprene startingcomposition derived from renewable resources comprises less than orabout 0.5 μg/L per compound for any compound in the composition thatinhibits the polymerization of isoprene.
 4. The method of claim 1wherein the polyisoprene produced from the isoprene starting material isa polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer hasf_(M) value which is greater than 0.9.
 5. The method of claim 1 whereinthe polymerization is a solution polymerization which is initiated withan anionic initiator.
 6. The method of claim 5 wherein the anionicinitiator is an alkyl lithium compound containing from 1 to 8 carbonatoms.
 7. The method of claim 6 wherein the polymerization is conductedin the presence of a polar modifier.
 8. The method of claim 1 whereinthe polymerization is allowed to continue until a conversion of at leastabout 85 percent is attained.
 9. The method of claim 1 wherein thepolymerization is initiated with a Ziegler Natta catalyst system. 10.The method of claim 9 wherein the Ziegler Natta catalyst system iscomprised of titanium tetrachloride and triethyl aluminum.
 11. A methodfor producing a copolymer of isoprene derived from renewable resourcescomprising: (a) initiating copolymerization of isoprene monomer with anon-isoprene molecule in an isoprene starting composition which isderived from renewable resources, wherein the isoprene startingcomposition derived from renewable resources comprises greater thanabout 2 mg of the isoprene monomer, at least one non-isoprene molecule,and one or more compounds selected from the group consisting of ethanol,acetone, C5 prenyl alcohols, isoprenoid compounds with 10 or more carbonatoms, methanol, acetaldehyde, methacrolein, methyl vinyl ketone,2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene,2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,and 2,3-cycloheptenolpyridine; (b) allowing the copolymerization of theisoprene monomer and the non-isoprene molecule to continue to producethe copolymer of isoprene; (c) terminating the copolymerization of theisoprene monomer and the non-isoprene molecule, and (d) recovering thecopolymer of isoprene.
 12. The method of claim 11, wherein the isoprenestarting composition derived from renewable resources comprises greaterthan or about 99.94% isoprene by weight compared to the total weight ofall C5 hydrocarbons in the composition.
 13. The method of claim 11,wherein the isoprene starting composition derived from renewableresources comprises less than or about 0.5 μg/L per compound for anycompound in the composition that inhibits the polymerization ofisoprene.
 14. The method of claim 11 wherein the non-isoprene moleculeis 1,3-butadiene.
 15. The method of claim 11 wherein the non-isoprenemolecule is styrene.
 16. The method of claim 11 wherein thepolymerization is a solution polymerization which is initiated with ananionic initiator.
 17. The method of claim 16 wherein the anionicinitiator is an alkyl lithium compound containing from 1 to 8 carbonatoms.
 18. The method of claim 17 wherein the polymerization isconducted in the presence of a polar modifier.
 19. The method of claim11 wherein the polymerization is allowed to continue until a conversionof at least about 85 percent is attained.
 20. The method of claim 11wherein the polymerization is initiated with a Ziegler Nana catalystsystem.